Glucose levels

ABSTRACT

Disclosed are methods and compositions for improving blood glucose levels in a subject in need thereof.

This application is a continuation application of U.S. application Ser.No. 11/166,499 filed Jun. 24, 2005, which is a continuation of U.S.application Ser. No. 10/795,552 filed Mar. 8, 2004 (now allowed), whichis a continuation of U.S. application Ser. No. 10/770,969 filed Feb. 2,2004, now U.S. Pat. No. 6,900,241, which is a divisional application ofU.S. application Ser. No. 10/127,817 filed Apr. 22, 2002 (now U.S. Pat.No. 6,998,417), which is a continuation of U.S. application Ser. No.09/717,893 filed Nov. 21, 2000, now U.S. Pat. No. 6,670,390, which is acontinuation of U.S. application Ser. No. 08/831,245 filed Apr. 2, 1997,now U.S. Pat. No. 6,297,273, which is a continuation-in-part of U.S.application Ser. No. 08/631,661 filed Apr. 2, 1996, now abandoned, eachof which is hereby incorporated herein by reference.

REFERENCE TO RELATED APPLICATION

Reference is made to the copending U.S. application Ser. No. 08/709,406,filed Sep. 6, 1996 (now U.S. Pat. No. 6,015,913), Ser. No. 08/631,661,filed Apr. 2, 1996 (now abandoned), and Ser. No. 08/317,226, filed Oct.3, 1994 (now U.S. Pat. No. 5,554,645) and PCT/US96/04497 (published asWO97/36597), each of which is incorporated herein by reference.

FIELD OF THE INVENTION

This invention relates to cocoa extracts and compounds therefrom such aspolyphenols preferably polyphenols enriched with procyanidins. Thisinvention also relates to methods for preparing such extracts andcompounds, as well as to uses for them; for instance, as antineoplasticagents, antioxidants, DNA topoisomerase II enzyme inhibitors,cyclo-oxygenase and/or lipoxygenase modulators, NO (Nitric Oxide) orNO-synthase modulators, as non-steroidal antiinflammatory agents,apoptosis modulators, platelet aggregation modulators, blood or in vivoglucose modulators, antimicrobials, and inhibitors of oxidative DNAdamage.

Documents are cited in this disclosure with a full citation for eachappearing thereat or in a References section at the end of thespecification, preceding the claims. These documents pertain to thefield of this invention; and, each document cited herein is herebyincorporated herein by reference.

BACKGROUND OF THE INVENTION

Polyphenols are an incredibly diverse group of compounds (Ferreira etal., 1992) which widely occur in a variety of plants, some of whichenter into the food chain. In some cases they represent an importantclass of compounds for the human diet. Although some of the polyphenolsare considered to be nonnutrative, interest in these compounds hasarisen because of their possible beneficial effects on health.

For instance, quercetin (a flavonoid) has been shown to possessanticarcinogenic activity in experimental animal studies (Deshner etal., 1991 and Kato et al., 1983). (+)-Catechin and (−)-epicatechin(flavan-3-ols) have been shown to inhibit Leukemia virus reversetranscriptase activity (Chu et al., 1992). Nobotanin (an oligomerichydrolyzable tannin) has also been shown to possess anti-tumor activity(Okuda et al., 1992). Statistical reports have also shown that stomachcancer mortality is significantly lower in the tea producing districtsof Japan. Epigallocatechin gallate has been reported to be thepharmacologically active material in green tea that inhibits mouse skintumors (Okuda et al., 1992). Ellagic acid has also been shown to possessanticarcinogen activity in various animal tumor models (Bukharta et al.,1992). Lastly, proanthocyanidin oligomers have been patented by theKikkoman Corporation for use as antimutagens. Indeed, the area ofphenolic compounds in foods and their modulation of tumor development inexperimental animal models has been recently presented at the 202^(nd)National Meeting of The American Chemical Society (Ho et al., 1992;Huang et al., 1992).

However, none of these reports teaches or suggests cocoa extracts orcompounds therefrom, any methods for preparing such extracts orcompounds therefrom, or, any uses for cocoa extracts or compoundstherefrom, as antineoplastic agents, antioxidants, DNA topoisomerase IIenzyme inhibitors, cyclo-oxygenase and/or lipoxygenase modulators, NO(Nitric Oxide) or NO-synthase modulators, as non-steroidalantiinflammatory agents, apoptosis modulators, platelet aggregationmodulators, blood or in vivo glucose modulators, antimicrobials, orinhibitors of oxidative DNA damage.

OBJECTS AND SUMMARY OF THE INVENTION

Since unfermented cocoa beans contain substantial levels of polyphenols,the present inventors considered it possible that similar activities ofand uses for cocoa extracts, e.g., compounds within cocoa, could berevealed by extracting such compounds from cocoa and screening theextracts for activity. The National Cancer Institute has screenedvarious Theobroma and Herrania species for anti-cancer activity as partof their massive natural product selection program. Low levels ofactivity were reported in some extracts of cocoa tissues, and the workwas not pursued. Thus, in the antineoplastic or anti-cancer art, cocoaand its extracts were not deemed to be useful; i.e., the teachings inthe antineoplastic or anti-cancer art lead the skilled artisan away fromemploying cocoa and its extracts as cancer therapy.

Since a number of analytical procedures were developed to study thecontributions of cocoa polyphenols to flavor development (Clapperton etal., 1992), the present inventors decided to apply analogous methods toprepare samples for anti-cancer screening, contrary to the knowledge inthe antineoplastic or anti-cancer art. Surprisingly, and contrary to theknowledge in the art, e.g., the National Cancer Institute screening, thepresent inventors discovered that cocoa polyphenol extracts whichcontain procyanidins, have significant utility as anti-cancer orantineoplastic agents.

Additionally, the inventors demonstrate that cocoa extracts containingprocyanidins and compounds from cocoa extracts have utility asantineoplastic agents, antioxidants, DNA topoisomerase II enzymeinhibitors, cyclo-oxygenase and/or lipoxygenase modulators, NO (NitricOxide) or NO-synthase modulators, as non-steroidal antiinflammatoryagents, apoptosis modulators, platelet aggregation modulators, blood orin vivo glucose modulators, antimicrobials, and inhibitors of oxidativeDNA damage.

It is an object of the present invention to provide a method forproducing cocoa extract and/or compounds therefrom.

It is another object of the invention to provide a cocoa extract and/orcompounds therefrom.

It is still another object of the present invention to provide apolymeric compound of the formula A_(n), wherein A is a monomer havingthe formula:

wherein

-   -   n is an integer from 2 to 18, such that there is at least one        terminal monomeric unit A, and a plurality of additional        monomeric units;    -   R is 3-(α)-OH, 3-(β)-OH, 3-(α)-O-sugar, or 3-(β)-O-sugar;        bonding between adjacent monomers takes place at positions 4, 6        or 8; a bond of an additional monomeric unit in position 4 has α        or β stereochemistry;    -   X, Y and Z are selected from the group consisting of monomeric        unit A, hydrogen, and a sugar, with the provisos that as to the        at least one terminal monomeric unit, bonding of the additional        monomeric unit thereto is at position 4 and Y=Z=hydrogen; the        sugar is optionally substituted with a phenolic moiety at any        position, for instance, via an ester bond, and pharmaceutically        acceptable salts or derivatives thereof (including oxidation        products).

It is still a further object of the present invention to provide apolymeric compound of the formula A_(n), wherein A is a monomer havingthe formula:

wherein

-   -   n is an integer from 2 to 18, e.g., 3 to 18;    -   R is 3-(α)-OH, 3-(β)-OH, 3-(α)-O-sugar, or 3-(β)-O-sugar;        adjacent monomers bind at position 4 by (4→6) or (4→8);    -   each of X, Y and Z is H, a sugar or an adjacent monomer, with        the provisos that if X and Y are adjacent monomers, Z is H or        sugar and if X and Z are adjacent monomers, Y is H or sugar, and        that as to at least one of the two terminal monomers, bonding of        the adjacent monomer is at position 4 and optionally,        Y=Z=hydrogen;    -   a bond at position 4 has α or β stereochemistry;

the sugar is optionally substituted with a phenolic moiety at anyposition, for instance, via an ester bond, and pharmaceuticallyacceptable salts or derivatives thereof (including oxidation products).

It is another object of the invention to provide an antioxidantcomposition.

It is another object of the invention to demonstrate inhibition of DNAtopoisomerase II enzyme activity.

It is yet another object of the present invention to provide a methodfor treating tumors or cancer.

It is still another object of the invention to provide an anti-cancer,anti-tumor or antineoplastic compositions.

It is still a further object of the invention to provide anantimicrobial composition.

It is yet another object of the invention to provide a cyclo-oxygenaseand/or lipoxygenase modulating composition.

It is still another object of the invention to provide an NO orNO-synthase-modulating composition.

It is a further object of the invention to provide a non-steroidalantiinflammatory composition.

It is another object of the invention to provide a blood or in vivoglucose-modulating composition.

It is yet a further object of the invention to provide a method fortreating a patient with an antineoplastic, antioxidant, antimicrobial,cyclo-oxygenase and/or lipoxygenase modulating or NO or NO-synthasemodulating non-steroidal antiinflammatory modulating and/or blood or invivo glucose-modulating composition.

It is an additional object of the invention to provide compositions andmethods for inhibiting oxidative DNA damage.

It is yet an additional object of the invention to provide compositionsand methods for platelet aggregation modulation.

It is still a further object of the invention to provide compositionsand methods for apoptosis modulation.

It is a further object of the invention to provide a method for makingany of the aforementioned compositions.

And, it is an object of the invention to provide a kit for use in theaforementioned methods or for preparing the aforementioned compositions.

It has been surprisingly discovered that cocoa extract, and compoundstherefrom, have anti-tumor, anti-cancer or antineoplastic activity or,is an antioxidant composition or, inhibits DNA topoisomerase II enzymeactivity or, is an antimicrobial or, is a cyclo-oxygenase and/orlipoxygenase modulator or, is a NO or NO-synthase modulator, is anon-steroidal antiinflammatory agent, apoptosis modulator, plateletaggregation modulator or, is a blood or in vivo glucose modulator, or isan inhibitor of oxidative DNA damage.

Accordingly, the present invention provides a substantially pure cocoaextract and compounds therefrom. The extract or compounds preferablycomprises polyphenol(s) such as polyphenol(s) enriched with cocoaprocyanidin(s), such as polyphenols of at least one cocoa procyanidinselected from (−) epicatechin, (+) catechin, procyanidin B-2,procyanidin oligomers 2 through 18, e.g., 3 through 18, such as 2through 12 or 3 through 12, preferably 2 through 5 or 4 through 12, morepreferably 3 through 12, and most preferably 5 through 12, procyanidinB-5, procyanidin A-2 and procyanidin C-1.

The present invention also provides an anti-tumor, anti-cancer orantineoplastic or antioxidant or DNA topoisomerase II inhibitor, orantimicrobial, or cyclo-oxygenase and/or lipoxygenase modulator, or anNO or NO-synthase modulator, nonsteroidal antiinflammatory agent,apoptosis modulator, platelet aggregation modulator, blood or in vivoglucose modulator, or oxidative DNA damage inhibitory compositioncomprising a substantially pure cocoa extract or compound therefrom orsynthetic cocoa polyphenol(s) such as polyphenol(s) enriched withprocyanidin(s) and a suitable carrier, e.g., a pharmaceutically,veterinary or food science acceptable carrier. The extract or compoundtherefrom preferably comprises cocoa procyanidin(s). The cocoa extractor compounds therefrom is preferably obtained by a process comprisingreducing cocoa beans to powder, defatting the powder and, extracting andpurifying active compound(s) from the powder.

The present invention further comprehends a method for treating apatient in need of treatment with an anti-tumor, anti-cancer, orantineoplastic agent or an antioxidant, or a DNA topoisomerase IIinhibitor, or antimicrobial, or cyclo-oxygenase and/or lipoxygenasemodulator, or an NO or NO-synthase modulator, non-steroidalantiinflammatory agent, apoptosis modulator, platelet aggregationmodulator, blood or in vivo glucose modulator or inhibitor of oxidativeDNA damage, comprising administering to the patient a compositioncomprising an effective quantity of a substantially pure cocoa extractor compound therefrom or synthetic cocoa polyphenol(s) or procyanidin(s)and a carrier, e.g., a pharmaceutically, veterinary or food scienceacceptable carrier. The cocoa extract or compound therefrom can be cocoaprocyanidin(s); and, is preferably obtained by reducing cocoa beans topowder, defatting the powder and, extracting and purifying activecompound(s) from the powder.

Additionally, the present invention provides a kit for treating apatient in need of treatment with an anti-tumor, anti-cancer, orantineoplastic agent or antioxidant or DNA topoisomerase II inhibitor,or antimicrobial, or cyclo-oxygenase and/or lipoxygenase modulator, oran NO or NO-synthase modulator, non-steroidal antiinflammatory agent,apoptosis modulator, platelet aggregation modulator inhibitor ofoxidative DNA damage, or blood or in vivo glucose modulator comprising asubstantially pure cocoa extract or compounds therefrom or syntheticcocoa polyphenol(s) or procyanidin(s) and a suitable carrier, e.g., apharmaceutically, veterinary or food science acceptable carrier, foradmixture with the extract or compound therefrom or syntheticpolyphenol(s) or procyanidin(s).

The present invention provides compounds as illustrated in FIGS. 38A to38P and 39A to 39AA; and linkages of 4→6 and 4→8 are presentlypreferred.

The invention even further encompasses food preservation or preparationcompositions comprising an inventive compound, and methods for preparingor preserving food by adding the composition to food.

And, the invention still further encompasses a DNA topoisomerase IIinhibitor comprising an inventive compound and a suitable carrier ordiluent, and methods for treating a patient in need of such treatment byadministration of the composition.

Considering broadly the aforementioned embodiments involving cocoaextracts, the invention also includes such embodiments wherein aninventive compound is used instead of or as the cocoa extracts. Thus,the invention comprehends kits, methods, and compositions analogous tothose above-stated with regard to cocoa extracts and with an inventivecompound.

These and other objects and embodiments are disclosed or will be obviousfrom the following Detailed Description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following Detailed Description will be better understood byreference to the accompanying drawings wherein:

FIG. 1 shows a representative gel permeation chromatogram from thefractionation of crude cocoa procyanidins;

FIG. 2A shows a representative reverse-phase HPLC chromatogram showingthe separation (elution profile) of cocoa procyanidins extracted fromunfermented cocoa;

FIG. 2B shows a representative normal phase HPLC separation of cocoaprocyanidins extracted from unfermented cocoa;

FIG. 3 shows several representative procyanidin structures;

FIGS. 4A-4E show representative HPLC chromatograms of five fractionsemployed in screening for anti-cancer or antineoplastic activity;

FIGS. 5 and 6A-6D show the dose-response relationship between cocoaextracts and cancer cells ACHN (FIG. 5) and PC-3 (FIGS. 6A-6D)(fractional survival vs. dose, 1 μg/mL); M&M2 F4/92, M&MA+E U12P1,M&MB+E Y192P1, M&MC+E U12P2, M&MD+E U12P2;

FIGS. 7A to 7H show the typical dose response relationships betweencocoa procyanidin fractions A, B, C, D, E, A+B, A+E, and A+D, and thePC-3 cell line (fractional survival vs. dose, μg/mL); MM-1A 0212P3, MM-1B 0162P1, MM-1 C 0122P3, MM-1 D 0122P3, MM-1 E 0292P8, MM-1 A/B 0292P6,MM-1 A/E 0292P6, MM-1 A/D 0292P6;

FIGS. 8A to 8H show the typical dose response relationships betweencocoa procyanidin fractions A, B, C, D, E, A+B, B+E, and D+E and the KBNasopharyngeal/HeLa cell line (fractional survival vs. dose, μg/mL);MM-1A092K3, MM-1 B 0212K5, MM-1 C 0162K3, MM-1 D 0212K5, MM-1 E 0292K5,MM-1 A/B 0292K3, MM-1 B/E 0292K4, MM-1 D/E 0292K5;

FIGS. 9A to 9H show the typical dose response relationship between cocoaprocyanidin fractions A, B, C, D, E, B+D, A+E and D+E and the HCT-116cell line (fractional survival vs. dose, μg/mL); MM-1 C 0192H5, D0192H5, E 0192H5, MM-1 B&D 0262H2, A/E 0262H3, MM-1 D&E 0262H1;

FIGS. 10A to 10H show typical dose response relationships between cocoaprocyanidin fractions A, B, C, D, E, B+D, C+D and A+E and the ACHN renalcell line (fractional survival vs. dose, μg/mL); MM-1 A 092A5, MM-1 B092A5, MM-1 C 0192A7, MM-1 D 0192A7, M&M1 E 0192A7, MM-1 B&D 0302A6,MM-1 C&D 0302A6, MM-1 A&E 0262A6;

FIGS. 11A to 11H show typical dose response relationships between cocoaprocyanidin fractions A, B, C, D, E, A+E, B+E and C+E and the A-549 lungcell line (fractional survival vs. dose, μg/mL); MM-1 A 019258, MM-1 B09256, MM-1 C 019259, MM-1 D 019258, MM-1 E 019258, A/E 026254, MM-1 B&E030255, MM-1 C&EN6255;

FIGS. 12A to 12H show typical dose response relationships between cocoaprocyanidin fractions A, B, C, D, E, B+C, C+D and D+E and the SK-5melanoma cell line (fractional survival vs. dose μg/mL); MM-1 A 0212S4,MM-1 B 0212S4, MM-1 C 0212S4, MM-1 D 0212S4, MM-1 E N32S1, MM-1 B&CN32S2, MM-1 C&D N32S3, MM-1 D&E N32S3;

FIGS. 13A to 13H show typical dose response relationships between cocoaprocyanidin fractions A, B, C, D, E, B+C, C+E, and D+E and the MCF-7breast cell line (fractional survival vs. dose, μg/mL); MM-1 A N22M4,MM-1 B N22M4, MM-1 C N22M4, MM-1 D N22M3, MM-1 E 0302M2, MM-1 B/C0302M4, MM-1 C&E N22M3, MM-1 D&E N22M3;

FIG. 14 shows typical dose response relationships for cocoa procyanidin(particularly fraction D) and the CCRF-CEM T-cell leukemia cell line(cells/mL vs. days of growth; open circle is control, darkened circle is125 μg fraction D, open inverted triangle is 250 μg fraction D, darkenedinverted triangle is 500 μg fraction D);

FIG. 15A shows a comparison of the XTT and Crystal Violet cytotoxicityassays against MCF-7 p168 breast cancer cells treated with fraction D+E(open circle is XTT and darkened circle is Crystal Violet);

FIG. 15B shows a typical dose response curve obtained from MDA MB231breast cell line treated with varying levels of crude polyphenolsobtained from UIT-1 cocoa genotype (absorbance (540 nm) vs. Days; opencircle is control, darkened circle is vehicle, open inverted triangle is250 μg/mL, darkened inverted triangle is 100 μg/mL, open square is 10μg/mL; absorbance of 2.0 is maximum of plate reader and may not benecessarily representative of cell number);

FIG. 15C shows a typical dose response curve obtained from PC-3 prostatecancer cell line treated with varying levels of crude polyphenolsobtained from UIT-1 cocoa genotype (absorbance (540 nm) vs. Days; opencircle is control, darkened circle is vehicle, open inverted triangle is250 μg/mL, darkened inverted triangle is 100 μg/mL and open square is 10μg/mL);

FIG. 15D shows a typical dose-response curve obtained from MCF-7 p168breast cancer cell line treated with varying levels of crude polyphenolsobtained from UIT-1 cocoa genotype (absorbance (540 nm) vs. Days; opencircle is control, darkened circle is vehicle, open inverted triangle is250 μg/mL, darkened inverted triangle is 100 μg/mL, open square is 10μg/mL, darkened square is 1 μg/mL; absorbance of 2.0 is maximum of platereader and may not be necessarily representative of cell number);

FIG. 15E shows a typical dose response curve obtained from Hela cervicalcancer cell line treated with varying levels of crude polyphenolsobtained from UIT-1 cocoa genotype (absorbance (540 nm) vs. Days; opencircle is control, darkened circle is vehicle, open inverted triangle is250 μg/mL, darkened inverted triangle is 100 μg/mL, open square is 10μg/mL; absorbance of 2.0 is maximum of plate reader and may not benecessarily representative of cell number);

FIG. 15F shows cytotoxic effects against Hela cervical cancer cell linetreated with different cocoa polyphenol fractions (absorbance (540 nm)vs. Days; open circle is 100 μg/mL fractions A-E, darkened circle is 100μg/mL fractions A-C, open inverted triangle is 100 μg/mL fractions D&E;absorbance of 2.0 is maximum of plate reader and not representative ofcell number);

FIG. 15G shows cytotoxic effects at 100 ul/mL against SKBR-3 breastcancer cell line treated with different cocoa polyphenol fractions(absorbance (540 nm) vs. Days; open circle is fractions A-E, darkenedcircle is fractions A-C, open inverted triangle is fractions D&E);

FIG. 15H shows typical dose-response relationships between cocoaprocyanidin fraction D+E on Hela cells (absorbance (540 nm) vs. Days;open circle is control, darkened circle is 100 μg/mL, open invertedtriangle is 75 μg/mL, darkened inverted triangle is 50 μg/mL, opensquare is 25 μg/mL, darkened square is 10 μg/mL; absorbance of 2.0 ismaximum of plate reader and is not representative of cell number);

FIG. 15I shows typical dose-response relationship between cocoaprocyanidin fraction D+E on SKBR-3 cells (absorbance (540 nm) vs. Days;open circle is control, darkened circle is 100 μg/mL, open invertedtriangle is 75 μg/mL, darkened inverted triangle is 50 μg/mL, opensquare is 25 μg/mL, darkened square is 10 μg/mL);

FIG. 15J shows typical dose-response relationships between cocoaprocyanidin fraction D+E on Hela cells using the Soft Agar Cloning assay(bar chart; number of colonies vs. control, 1, 10, 50, and 100 μg/mL);

FIG. 15K shows the growth inhibition of Hela cells when treated withcrude polyphenol extracts obtained from eight different cocoa genotypes(% control vs. concentration, μg/mL; open circle is C-1, darkened circleis C-2, open inverted triangle is C-3, darkened inverted triangle isC-4, open square is C-5, darkened square is C-6, open triangle is C-7,darkened triangle is C-8; C-1=UF-12: horti race=Trinitario anddescription is crude extracts of UF-12 (Brazil) cocoa polyphenols(decaffeinated/detheobrominated); C-2=NA-33: horti race=Forastero anddescription is crude extracts of NA-33 (Brazil) cocoa polyphenols(decaffeinated/detheobrominated); C-3=EEG-48: horti race=Forastero anddescription is crude extracts of EEG-48 (Brazil) cocoa polyphenols(decaffeinated/detheobrominated); C-4=unknown: horti race=Forastero anddescription is crude extracts of unknown (W. African) cocoa polyphenols(decaffeinated/detheobrominated); C-5=UF-613: horti race=Trinitario anddescription is crude extracts of UF-613 (Brazil) cocoa polyphenols(decaffeinated/detheobrominated); C-6=ICS-100: horti race=Trinitario (toNicaraguan Criollo ancestor) and description is crude extracts ofICS-100 (Brazil) cocoa polyphenols (decaffeinated/detheobrominated);C-7=ICS-139: horti race=Trinitario (Nicaraguan Criollo ancestor) anddescription is crude extracts of ICS-139 (Brazil) cocoa polyphenols(decaffeinated/detheobrominated); C-8=UIT-1: horti race=Trinitario anddescription is crude extracts of UIT-1 (Malaysia) cocoa polyphenols(decaffeinated/detheobrominated);

FIG. 15L shows the growth inhibition of Hela cells when treated withcrude polyphenol extracts obtained from fermented cocoa beans and driedcocoa beans (stages throughout fermentation and sun drying; % controlvs. concentration, μg/mL; open circle is day zero fraction, darkenedcircle is day 1 fraction, open inverted triangle is day 2 fraction,darkened inverted triangle is day 3 fraction, open square is day 4fraction and darkened square is day 9 fraction);

FIG. 15M shows the effect of enzymatically oxidized cocoa procyanidinsagainst Hela cells (dose response for polyphenol oxidase treated crudecocoa polyphenol; % control vs. concentration, μg/mL; darkened square iscrude UIT-1 (with caffeine and theobromine), open circle crude UIT-1(without caffeine and theobromine) and darkened circle is crude UIT-1(polyphenol oxidase catalyzed);

FIG. 15N shows a representative semi-preparative reverse phase HPLCseparation for combined cocoa procyanidin fractions D and E;

FIG. 15O shows a representative normal phase semi-preparative HPLCseparation of a crude cocoa polyphenol extract;

FIG. 16 shows typical Rancimat Oxidation curves for cocoa procyanidinextract and fractions in comparison to the synthetic antioxidants BHAand BHT (arbitrary units vs. time; dotted line and cross (+) is BHA andBHT; * is D-E; x is crude; open square is A-C; and open diamond iscontrol);

FIG. 17 shows a typical Agarose Gel indicating inhibition oftopoisomerase II catalyzed decatenation of kinetoplast DNA by cocoaprocyanidin fractions (Lane 1 contains 0.5 μg of marker (M)monomer-length kinetoplast DNA circles; Lanes 2 and 20 containkinetoplast DNA that was incubated with Topoisomerase II in the presenceof 4% DMSO, but in the absence of any cocoa procyanidins. (Control-C);Lanes 3 and 4 contain kinetoplast DNA that was incubated withTopoisomerase II in the presence of 0.5 and 5.0 μg/mL cocoa procyanidinfraction A; Lanes 5 and 6 contain kinetoplast DNA that was incubatedwith Topoisomerase II in the presence of 0.5 and 5.0 μg/mL cocoaprocyanidin fraction B; Lanes 7, 8, 9, 13, 14 and 15 are replicates ofkinetoplast DNA that was incubated with Topoisomerase II in the presenceof 0.05, 0.5 and 5.0 μg/mL cocoa procyanidin fraction D; Lanes 10, 11,12, 16, 17 and 18 are replicates of kinetoplast DNA that was incubatedwith Topoisomerase II in the presence of 0.05, 0.5, and 5.0 μg/mL cocoaprocyanidin fraction E; Lane 19 is a replicate of kinetoplast DNA thatwas incubated with Topoisomerase II in the presence of 5.0 μg/mL cocoaprocyanidin fraction E);

FIG. 18 shows dose response relationships of cocoa procyanidin fractionD against DNA repair competent and deficient cell lines (fractionalsurvival vs. μg/mL; left side xrs-6 DNA Deficient Repair Cell Line, MM-1D D282X1; right side BR1 Competent DNA Repair Cell Line, MM-1 D D282B1);

FIG. 19 shows the dose-response curves for Adriamycin resistant MCF-7cells in comparison to a MCF-7 p168 parental cell line when treated withcocoa fraction D+E (% control vs. concentration, μg/mL; open circle isMCF-7 p168; darkened circle is MCF-7 ADR);

FIGS. 20A and B show the dose-response effects on Hela and SKBR-3 cellswhen treated at 100 μg/mL and 25 μg/mL levels of twelve fractionsprepared by Normal phase semi-preparative HPLC (bar chart, % control vs.control and fractions 1-12);

FIGS. 21A, 21B and 21C show a normal phase HPLC separation of crude,enriched and purified pentamers from cocoa extract;

FIGS. 22A, B and C show MALDI-TOF/MS of pentamer enriched procyanidins,and of Fractions A-C and of Fractions D-E, respectively;

FIG. 23A shows an elution profile of oligomeric procyanidins purified bymodified semi-preparative HPLC;

FIG. 23B shows an elution profile of a trimer procyanidin by modifiedsemi-preparative HPLC;

FIGS. 24A-D each show energy minimized structures of all (4-8) linkedpentamers based on the structure of epicatechin;

FIG. 25A shows relative fluorescence of epicatechin upon thiolysis withbenzylmercapten;

FIG. 25B shows relative fluorescence of catechin upon thiolysis withbenzylmercapten;

FIG. 25C shows relative fluorescence of dimers (B2 and B5) uponthiolysis with benzylmercapten;

FIG. 26A shows relative fluorescence of dimer upon thiolysis;

FIG. 26B shows relative fluorescence of B5 dimer upon thiolysis of dimerand subsequent desulphurization;

FIG. 27A shows the relative tumor volume during treatment of MDA MB 231nude mouse model treated with pentamer;

FIG. 27B shows the relative survival curve of pentamer treated MDA 231nude mouse model;

FIG. 28 shows the elution profile from halogen-free analyticalseparation of acetone extract of procyanidins from cocoa extract;

FIGS. 29A, 29B and 29C show the effect of pore size of stationary phasefor normal phase HPLC separation of procyanidins;

FIG. 30A shows the substrate utilization during fermentation of cocoabeans;

FIG. 30B shows the metabolite production during fermentation;

FIG. 30C shows the plate counts during fermentation of cocoa beans;

FIG. 30D shows the relative concentrations of each component infermented solutions of cocoa beans;

FIG. 31 shows the acetylcholine-induced relaxation of NO-relatedphenylephrine-precontracted rat aorta;

FIG. 32 shows the blood glucose tolerance profiles from various testmixtures;

FIGS. 33A-B show the effects of indomethacin on COX-1 and COX-2activities;

FIGS. 34A-B show the correlation between the degree of polymerizationand IC₅₀ vs. COX-1/COX-2 (μM);

FIG. 35 shows the correlation between the effects of compounds on COX-1and COX-2 activities expressed as μM;

FIGS. 36A-V show the IC₅₀ values (μM) of samples containing procyanidinswith COX-1/COX-2;

FIG. 37 shows the purification scheme for the isolation of procyanidinsfrom cocoa;

FIG. 38A to 38P shows the preferred structures of the pentamer;

FIGS. 39A-AA show a library of stereoisomers of pentamers;

FIGS. 40A-B show 70 minute gradients for normal phase HPLC separation ofprocyanidins, detected by UV and fluorescence, respectively;

FIGS. 41A-B show 30 minute gradients for normal phase HPLC separation ofprocyanidins, detected by UV and fluorescence, respectively;

FIG. 42 shows a preparation normal phase HPLC separation ofprocyanidins;

FIGS. 43A-G show CD (circular dichroism) spectra of procyanidin dimers,trimers, tetramers, pentamers, hexamers, heptamers and octamers,respectively;

FIG. 44A shows the structure and ¹H/¹³C NMR data for epicatechin;

FIGS. 44B-F show the APT, COSY, XHCORR, ¹H and ¹³C NMR spectra forepicatechin;

FIG. 45A shows the structure and ¹H/¹³C NMR data for catechin;

FIGS. 45B-E show the ¹H, APT, XHCORR and COSY NMR spectra for catechin;

FIG. 46A shows the structure and ¹H/¹³C NMR data for B2 dimer;

FIGS. 46B-G show the ¹³C, APT, ¹H, HMQC, COSY and HOHAHA NMR spectra forthe B2 dimer;

FIG. 47A shows the structure and ¹H/¹³C NMR data for B5 dimer;

FIGS. 47B-G show the ¹H, ¹³C, APT, COSY, HMQC and HOHAHA NMR spectra forB5 dimer;

FIGS. 48A-D show the ¹H, COSY, HMQC and HOHAHA NMR spectra forepicatechin/catechin trimer;

FIGS. 49A-D show the ¹H, COSY, HMQC and HOHAHA NMR spectra forepicatechin trimer;

FIGS. 50A and B show the effects of cocoa procyanidin fraction A and C,respectively, on blood pressure; blood pressure levels decreased by21.43% within 1 minute after administration of fraction A, and returnedto normal after 15 minutes, while blood pressure decreased by 50.5%within 1 minute after administration of fraction C, and returned tonormal after 5 minutes;

FIG. 51 shows the effect of cocoa procyanidin fractions on arterialblood pressure in anesthetized guinea pigs;

FIG. 53 shows the effect of bradykinin on NO production by HUVEC;

FIG. 54 shows the effect of cocoa procyanidin fractions on macrophage NOproduction by HUVEC;

FIG. 55 shows the effect of cocoa extracts on LPS-induced nitriteproduction by γ-interferon-primed monocytes/macrophages. Cocoa extractsA, C, D enhanced the LPS-induced nitrite production byγ-interferon-primed monocytes/macrophages up to respectively 12.8, 5.2and 26.8 μM nitrite. LPS stimulated monocytes/macrophages cultured inthe absence of cocoa extract produced 4 μM nitrite;

FIG. 56 shows the direct effect of cocoa extracts on nitrite productionby monocytes/macrophages. Only extract C at 100 μg/ml induced production(24 μM) by monocytes/macrophages whereas the basal production by thesecells was undetectable. The other extract did not evoke NO production,as assessed by the measure of nitrite in supernatants;

FIG. 57 shows a micellar electrokinetic capillary chromatographicseparation of cocoa procyanidin oligomers;

FIG. 58 A-F show MALDI-TOF mass spectra for Cu⁺²—, Zn⁺²—, Fe⁺²—, Fe⁺³—,Ca⁺²—, and Mg⁺²— ions, respectively, complexed to a trimer;

FIG. 59 shows a MALDI-TOF mass spectrum of cocoa procyanidin oligomers(tetramers to octadecamers);

FIG. 60 shows the dose-response relationship of cocoa procyanidinoligomers and the feline FeA lymphoblastoid cell line producing leukemiavirus;

FIG. 61 shows the dose-response relationship of cocoa procyanidinoligomers and the feline CRFK normal kidney cell line;

FIG. 62 shows the dose-response relationship of cocoa procyanidinoligomers and the canine MDCK normal kidney line;

FIG. 63 shows the dose-response relationship between cocoa procyanidinoligomers and the canine GH normal kidney cell line;

FIG. 64 shows time-temperature effects on hexamer hydrolysis; and

FIG. 65 shows time-temperature effects on trimer formation.

DETAILED DESCRIPTION

Compounds of the Invention

As discussed above, it has now been surprisingly found that cocoaextracts or compounds derived therefrom exhibit anti-cancer, anti-tumoror antineoplastic activity, antioxidant activity, inhibit DNAtopoisomerase II enzyme and oxidative damage to DNA, and haveantimicrobial, cyclo-oxygenase and/or lipoxygenase, NO or NO-synthase,apoptosis, platelet aggregation and blood or in vivo glucose-modulatingactivities, as well as efficacy as a non-steroidal antiinflammatoryagent.

The extracts, compounds or combination of compounds derived therefromare generally prepared by reducing cocoa beans to a powder, defattingthe powder, and extracting and purifying the active compound(s) from thedefatted powder. The powder can be prepared by freeze-drying the cocoabeans and pulp, depulping and dehulling the freeze-dried cocoa beans andgrinding the dehulled beans. The extraction of active compound(s) can beby solvent extraction techniques. The extracts comprising the activecompounds can be purified, e.g., to be substantially pure, for instance,by gel permeation chromatography or by preparative High PerformanceLiquid Chromatography (HPLC) techniques or by a combination of suchtechniques.

With reference to the isolation and purification of the compounds of theinvention derived from cocoa, it will be understood that any species ofTheobroma, Herrania or inter- and intra-species crosses thereof may beemployed. In this regard, reference is made to Schultes, “Synopsis ofHerrania,” Journal of the Arnold Arboretum, Vol. XXXIX, pp. 217 to 278,plus plates I to XVII (1985), Cuatrecasas, “Cocoa and Its Allies, ATaxonomic Revision of the Genus Theobroma,” Bulletin of the UnitedStates National Museum, Vol. 35, part 6, pp. 379 to 613, plus plates 1to 11 (Smithsonian Institution, 1964), and Addison, et al.,“Observations on the Species of the Genus Theobroma Which Occurs in theAmazon,” Bol. Tech. Inst. Agronomico de Nortes, 25(3) (1951).

Additionally, Example 25 lists the heretofore never reportedconcentrations of the inventive compounds found in Theobroma andHerrania species and their inter- and intra-species crosses; and Example25 also describes methods of modulating the amounts of the inventivecompounds which may be obtained from cocoa by manipulating cocoafermentation conditions.

An outline of the purification protocol utilized in the isolation ofsubstantially pure procyanidins is shown in FIG. 37. Steps 1 and 2 ofthe purification scheme are described in Examples 1 and 2; steps 3 and 4are described in Examples 3, 13 and 23; step 5 is described in Examples4 and 14; and step 6 is described in Examples 4, 14 and 16. The skilledartisan would appreciate and envision modifications in the purificationscheme outlined in FIG. 37 to obtain the active compounds withoutdeparting from the spirit or scope thereof and without undueexperimentation.

The extracts, compounds and combinations of compounds derived therefromhaving activity, without wishing to necessarily be bound by anyparticular theory, have been identified as cocoa polyphenol(s), such asprocyanidins. These cocoa procyanidins have significant anti-cancer,anti-tumor or antineoplastic activity; antioxidant activity; inhibit DNAtopoisomerase II enzyme and oxidative damage to DNA; possessantimicrobial activity; have the ability to modulate cyclo-oxygenaseand/or lipoxygenase, NO or NO-synthase, apoptosis, platelet aggregationand blood or in vivo glucose, and have efficacy as non-steroidalantiinflammatory agents.

The present invention provides a compound of the formula:

wherein:

-   -   n is an integer from 2 to 18, e.g., 3 to 12, such that there is        a first monomeric unit A, and a plurality of other monomeric        units;    -   R is 3-(α)-OH, 3-(β)-OH, 3-(α)-O-sugar, or 3-(β)-O-sugar;    -   position 4 is alpha or beta stereochemistry;    -   X, Y and Z represent positions for bonding between monomeric        units, with the provisos that as to the first monomeric unit,        bonding of another monomeric unit thereto is at position 4 and        Y=Z=hydrogen, and, that when not for bonding monomeric units, X,        Y and Z are hydrogen, or Z, Y are sugar and X is hydrogen, or X        is alpha or beta sugar and Z, Y are hydrogen, or combinations        thereof. The compound can have n as 5 to 12, and certain        preferred compounds have n as 5. The sugar can be selected from        the group consisting of glucose, galactose, xylose, rhamnose,        and arabinose. The sugar of any or all of R, X, Y and Z can        optionally be substituted with a phenolic moiety via an ester        bond.

Thus, the invention can provide a compound of the formula

wherein:

-   -   n is an integer from 2 to 18, e.g., 3 to 12, advantageously 5 to        12, and preferably n is 5, such that there is a first monomeric        unit A,    -   and a plurality of other monomeric units of A;    -   R is 3-(α)-OH, 3-(β)-OH, 3-(α)-O-sugar, or 3-(β)-O-sugar;    -   position 4 is alpha or beta stereochemistry;    -   X, Y and Z represent positions for bonding between monomeric        units, with the provisos that as to the first monomeric unit,        bonding of another monomeric unit thereto is at position 4 and        Y=Z=hydrogen, and, that when not for bonding monomeric units, X,        Y and Z are hydrogen or Z, Y are sugar and X is hydrogen, or X        is alpha or beta sugar and Z and Y are hydrogen, or combinations        thereof; and said sugar is optionally substituted with a        phenolic moiety via an ester bond.

Accordingly, the present invention provides a polymeric compound of theformula A_(n), wherein A is a monomer having the formula:

wherein

-   -   n is an integer from 2 to 18, such that there is at least one        terminal monomeric unit A, and at least one or a plurality of        additional monomeric units;    -   R is 3-(α)-OH, 3-(β)-OH, 3-(α)-O-sugar, or 3-(β)-O-sugar;    -   bonding between adjacent monomers takes place at positions 4, 6        or 8;    -   a bond of an additional monomeric unit in position 4 has α or β        stereochemistry;    -   X, Y and Z are selected from the group consisting of monomeric        unit A, hydrogen, and a sugar, with the provisos that as to the        at least one terminal monomeric unit, bonding of the additional        monomeric unit thereto (i.e., the bonding of the monomeric unit        adjacent the terminal monomeric unit) is at position 4 and        optionally, Y=Z=hydrogen;    -   the sugar is optionally substituted with a phenolic moiety at        any position, for instance via an ester bond, and        pharmaceutically acceptable salts or derivatives thereof        (including oxidation products).    -   In preferred embodiments, n can be 3 to 18, 2 to 18, 3 to 12,        e.g., 5 to 12; and, advantageously, n is 5. The sugar is        selected from the group consisting of glucose, galactose,        xylose, rhamnose and arabinose. The sugar of any or all of R, X,        Y and Z can optionally be substituted at any position with a        phenolic moiety via an ester bond. The phenolic moiety is        selected from the group consisting of caffeic, cinnamic,        coumaric, ferulic, gallic, hydroxybenzoic and sinapic acids.

Additionally, the present invention provides a polymeric compound of theformula A_(n), wherein A is a monomer having the formula:

wherein

-   -   n is an integer from 2 to 18, e.g., 3 to 18, advantageously 3 to        12, e.g., 5 to 12, preferably n is 5;    -   R is 3-(α)-OH, 3-(β)-OH, 3-(α)-O-sugar, or 3-(β)-O-sugar;    -   adjacent monomers bind at position 4 by (4→6) or (4→8);    -   each of X, Y and Z is H, a sugar or an adjacent monomer, with        the provisos that if X and Y are adjacent monomers, Z is H or        sugar and if X and Z are adjacent monomers, Y is H or sugar, and        that as to at least one of the two terminal monomers, bonding of        the adjacent monomer is at position 4 and optionally,        Y=Z=hydrogen;    -   a bond at position 4 has α or β stereochemistry;    -   the sugar is optionally substituted with a phenolic moiety at        any position, for instance, via an ester bond, and        pharmaceutically acceptable salts or derivatives thereof        (including oxidation products).

With regard to the recitation of “at least one terminal monomeric unitA”, it will be understood that the inventive compounds have two terminalmonomeric units, and that the two terminal monomeric unit A may be thesame or different. Additionally, it will be understood that therecitation of “at least one terminal monomeric unit A” includesembodiments wherein the terminal monomeric unit A is referred to as a“first monomeric unit”, with the recitation of “first monomeric unit”relating to that monomer to which other monomeric units are added,resulting in a polymeric compound of the formula A_(n). Moreover, withregard to the at least one of the two terminal monomers, bonding of theadjacent monomer is at position 4 and optionally, Y=Z=hydrogen.

As to the recitation of the term “combinations thereof”, it will beunderstood that one or more of the inventive compounds may be usedsimultaneously, e.g., administered to a subject in need of treatment ina formulation comprising one or more inventive compounds.

The inventive compounds or combinations thereof display the utilitiesnoted above for cocoa extracts; and throughout the disclosure, the term“cocoa extract” may be substituted by compounds of the invention orcombinations thereof, such that it will be understood that the inventivecompounds or combinations thereof can be cocoa extracts.

The term “oligomer”, as used herein, refers to any compounds orcombinations thereof of the formula presented above, wherein n is 2through 18. When n is 2, the oligomer is termed a “dimer”; when n is 3,the oligomer is termed a “trimer”; when n is 4, the oligomer is termed a“tetramer”; when n is 5, the oligomer is termed a “pentamer”; andsimilar recitations may be designated for oligomers having n up to andincluding 18, such that when n is 18, the oligomer is termed an“octadecamer”.

The inventive compounds or combinations thereof can be isolated, e.g.,from a natural source such as any species of Theobroma, Herrania orinter- or intra-species crosses thereof; or, the inventive compounds orcombinations thereof can be purified, e.g., compounds or combinationsthereof can be substantially pure; for instance, purified to apparenthomogeneity. Purity is a relative concept, and the numerous Examplesdemonstrate isolation of inventive compounds or combinations thereof, aswell as purification thereof, such that by methods exemplified a skilledartisan can obtain a substantially pure inventive compound orcombination thereof, or purify them to apparent homogeneity (e.g.,purity by separate, distinct chromatographic peak). Considering theExamples (e.g., Example 37), a substantially pure compound orcombination of compounds is at least about 40% pure, e.g., at leastabout 50% pure, advantageously at least about 60% pure, e.g., at leastabout 70% pure, more advantageously at least about 75-80% pure,preferably, at least about 90% pure, more preferably greater than 90%pure, e.g., at least 90-95% pure, or even purer, such as greater than95% pure, e.g., 95-98% pure.

Further, examples of the monomeric units comprising the oligomers usedherein are (+)-catechin and (−)-epicatechin, abbreviated C and EC,respectively. The linkages between adjacent monomers are from position 4to position 6 or position 4 to position 8; and this linkage betweenposition 4 of a monomer and position 6 and 8 of the adjacent monomericunits is designated herein as (4→6) or (4→8). There are four possiblestereochemical linkages between position 4 of a monomer and position 6and 8 of the adjacent monomer; and the stereochemical linkages betweenmonomeric units is designated herein as (4α→6) or (4β→6) or (4α→8) or(4β→8). When C is linked to another C or EC, the linkages are designatedherein as (4α→6) or (4α→8). When EC is linked to another C or EC, thelinkages are designated herein as (4β→6) or (4β→8).

Examples of compounds eliciting the activities cited above includedimers, EC-(4β→8)-EC and EC-(4β→6)-EC, wherein EC-(4β→8)-EC ispreferred; trimers [EC-(4β→8)]₂-EC, [EC-(4β→8)]₂-C and [EC-(4β→6)]₂-EC,wherein [EC-(4β→8)]₂-EC is preferred; tetramers [EC-(4β→8)]₃-EC,[EC-(4β→8)]₃-C and [EC-(4β→8)]₂-EC-(4β→6)-C, wherein [EC-(4β→8)]₃-EC ispreferred; and pentamers [EC-(4β→8)]₄-EC, [EC-(4β→8)]₃-EC-(4β→6)-EC,[EC-(4β→8)]₃-EC-(4β→8)-C and [EC-(4β→8)]₃-EC-(4β→6)-C, wherein the3-position of the pentamer terminal monomeric unit is optionallyderivatized with a gallate or β-D-glucose; [EC-(4β→8)]₄-EC is preferred.

Additionally, compounds which elicit the activities cited above alsoinclude hexamers to dodecamers, examples of which are listed below:

A hexamer, wherein one monomer (C or EC) having linkages to anothermonomer (4β→8) or (4β→6) for EC linked to another EC or C, and (4α→8) or(4α→6) for C linked to another C or EC; followed by a (4β→8) linkage toa pentamer compound listed above, e.g., [EC-(4β→8)]₅-EC,[EC-(4β→8)]₄-EC-(4β→6)-EC, [EC-(4β→8)]₄-EC-(4β→8)-C, and[EC-(4β→8)]₄-EC-(4β→6)-C, wherein the 3-position of the hexamer terminalmonomeric unit is optionally derivatized with a gallate or aβ-D-glucose; in a preferred embodiment, the hexamer is [EC-(4β→8)]₅-EC;

A heptamer, wherein any combination of two monomers (C and/or EC) havinglinkages to one another (4β→8) or (4β→6) for EC linked to another EC orC, and (4α→8) or (4α→6) for C linked to another C or EC; followed by a(4β→8) linkage to a pentamer compound listed above, e.g.,[EC-(4β→8)]₆-EC, [EC-(4β→8)]₅-EC-(4β→6)-EC, [EC-(4β→8)]₅-EC-(4β→8)-C,and [EC-(4β→8)]₅-EC-(4β→6)-C, wherein the 3-position of the heptamerterminal monomeric unit is optionally derivatized with a gallate or aβ-D-glucose; in a preferred embodiment, the heptamer is [EC-(4β→8)]₆-EC;

An octamer, wherein any combination of three monomers (C and/or EC)having linkages to one another (4β→8) or (4β→6) for EC linked to anotherEC or C, and (4α→8) or (4α→6) for C linked to another C or EC; followedby a (4β→8) linkage to a pentamer compound listed above, e.g.,[EC-(4β→8)]₇-EC, [EC-(4β→8)]₆-EC-(4β→6)-EC, [EC-(4β→8)]₆-EC-(4β→8)-C,and [EC-(4β→8)]₆-EC-(4β→6)-C, wherein the 3-position of the octamerterminal monomeric unit is optionally derivatized with a gallate or aβ-D-glucose; in a preferred embodiment, the octamer is [EC-(4β→8)]₇-EC;

A nonamer, wherein any combination of four monomers (C and/or EC) havinglinkages to one another (4β→8) or (4β→6) for EC linked to another EC orC, and (4α→8) or (4α→6) for C linked to another C or EC; followed by a(4β→8) linkage to a pentamer compound listed above, e.g.,[EC-(4β→8)]₈-EC, [EC-(4β→8)]₇-EC-(4β→6)-EC, [EC-(4β→8)]₇-EC-(4β→8)-C,and [EC-(4β→8)]₇-EC-(4→→6)-C, wherein the 3-position of the nonamerterminal monomeric unit is optionally derivatized with a gallate or aβ-D-glucose; in a preferred embodiment, the nonamer is [EC-(4β→8)]₈-EC;

A decamer, wherein any combination of five monomers (C and/or EC) havinglinkages to one another (4β→8) or (4β→6) for EC linked to another EC orC, and (4α→8) or (4α→6) for C linked to another C or EC; followed by a(4β→8) linkage to a pentamer compound listed above, e.g.,[EC-(4β→8)]₉-EC, [EC-(4β→8)]₈-EC-(4β→6)-EC, [EC-(4β→8)]₈-EC-(4β→8)-C,and [EC-(4β→8)]₈-EC-(4β→6)-C, wherein the 3-position of the decamerterminal monomeric unit is optionally derivatized with a gallate or aβ-D-glucose; in a preferred embodiment, the decamer is [EC-(4β→8)]₉-EC;

An undecamer, wherein any combination of six monomers (C and/or EC)having linkages to one another (4β→8) or (4β→6) for EC linked to anotherEC or C, and (4α→8) or (4α→6) for C linked to another C or EC; followedby a (4β→8) linkage to a pentamer compound listed above, e.g.,[EC-(4β→8)]₁₀-EC, [EC-(4β→8)]₉-EC-(4β→6)-EC, [EC-(4β→8)]₉-EC-(4β→8)-C,and [EC-(4β→8)]₉-EC-(4β→6)-C, wherein the 3-position of the undecamerterminal monomeric unit is optionally derivatized with a gallate or aβ-D-glucose; in a preferred embodiment, the undecamer is[EC-(4β→8)]₁₀-EC; and

A dodecamer, wherein any combination of seven monomers (C and/or EC)having linkages to one another (4β→8) or (4β→6) for EC linked to anotherEC or C, and (4α→8) or (4α→6) for C linked to another C or EC; followedby a (4β→8) linkage to a pentamer compound listed above, e.g.,[EC-(4β→8)]₁₁-EC, [EC-(4β→8)]₁₀-EC-(4β→6)-EC, [EC-(4β→8)]₁₀-EC-(4β→8)-C,and [EC-(4β→8)]₁₀-EC-(4β→6)-C, wherein the 3-position of the dodecamerterminal monomeric unit is optionally derivatized with a gallate or aβ-D-glucose; in a preferred embodiment, the dodecamer is[EC-(4β→8)]₁₁-EC.

It will be understood from the detailed description that theaforementioned list is exemplary and provided as an illustrative sourceof several non-limiting examples of compounds of the invention, which isby no means an exhaustive list of the inventive compounds encompassed bythe present invention.

Examples 3A, 3B, 4, 14, 23, 24, 30 and 34 describe methods to separatethe compounds of the invention. Examples 13, 14A-D and 16 describemethods to purify the compounds of the invention. Examples 5, 15, 18,19, 20 and 29 describe methods to identify compounds of the invention.FIGS. 38A-P and 39A-AA illustrate a stereochemical library forrepresentative pentamers of the invention. Example 17 describes a methodto molecularly model the compounds of the invention. Example 36 providesevidence for higher oligomers in cocoa, wherein n is 13 to 18.

Furthermore, while the invention is described with respect to cocoaextracts preferably comprising cocoa procyanidins, from this disclosurethe skilled organic chemist will appreciate and envision syntheticroutes to obtain and/or prepare the active compounds (see e.g., Example11). Accordingly, the invention comprehends synthetic cocoa polyphenolsor procyanidins or their derivatives and/or their synthetic precursorswhich include, but are not limited to glycosides, gallates, esters, etc.and the like. That is, the inventive compounds can be prepared fromisolation from cocoa or from any species within the Theobroma orHerrania genera, as well as from synthetic routes; and derivatives andsynthetic precursors of the inventive compounds such as glycosides,gallates, esters, etc. are included in the inventive compounds.Derivatives can also include compounds of the above formulae wherein asugar or gallate moiety is on the terminal monomer at positions Y or Z,or a substituted sugar or gallate moiety is on the terminal monomer at Yor Z.

For example, Example 8, Method C describes the use of cocoa enzymes tooxidatively modify the compounds of the invention or combinationsthereof to elicit improved cytotoxicity (see FIG. 15M) against certaincancer cell lines. The invention includes the ability to enzymaticallymodify (e.g., cleavage or addition of a chemically significant moiety)the compounds of the invention, e.g., enzymatically with polyphenoloxidase, peroxidase, catalase combinations, and/or enzymes such ashydrolases, esterases, reductases, transferases, and the like and in anycombination, taking into account kinetic and thermodynamic factors (seealso Example 41 regarding hydrolysis).

With regard to the synthesis of the inventive compounds, the skilledartisan will be able to envision additional routes of synthesis, basedon this disclosure and the knowledge in the art, without undueexperimentation. For example, based upon a careful retrosyntheticanalysis of the polymeric compounds, as well as the monomers. Forinstance, given the phenolic character of the inventive compounds, theskilled artisan can utilize various methods of selectiveprotection/deprotection, coupled with organometallic additions, phenoliccouplings and photochemical reactions, e.g., in a convergent, linear orbiomimetic approach, or combinations thereof, together with standardreactions known to those well-versed in the art of synthetic organicchemistry, as additional synthetic methods for preparing the inventivecompounds, without undue experimentation. In this regard, reference ismade to W. Carruthers, Some Modern Methods of Organic Synthesis, 3^(rd)ed., Cambridge University Press, 1986, and J. March, Advanced OrganicChemistry, 3^(rd) ed., John Wiley & Sons, 1985, van Rensburg et al.,Chem. Comm., 24: 2705-2706 (Dec. 21, 1996), Ballenegger et al., (Zyma SA) European Patent 0096 007 B1, and documents in the References sectionbelow, all of which are hereby incorporated herein by reference.

UTILITIES OF COMPOUNDS OF THE INVENTION

With regard to the inventive compounds, it has been surprisingly foundthat the inventive compounds have discrete activities, and as such, theinventive compounds have broad applicability to the treatment of avariety of disease conditions, discussed hereinbelow.

COX/LOX-Associated Utilities

Atherosclerosis, the most prevalent of cardiovascular diseases, is theprinciple cause of heart attack, stroke and vascular circulationproblems. Atherosclerosis is a complex disease which involves many celltypes, biochemical events and molecular factors. There are severalaspects of this disease, its disease states and disease progressionwhich are distinguished by the interdependent consequences of LowDensity Lipoprotein (LDL) oxidation, cyclo-oxygenase (COX)/lipoxygenase(LOX) biochemistry and Nitric Oxide (NO) biochemistry.

Clinical studies have firmly established that the elevated plasmaconcentrations of LDL are associated with accelerated atherogenesis. Thecholesterol that accumulates in atherosclerotic lesions originateprimarily in plasma lipoproteins, including LDL. The oxidation of LDL isa critical event in the initiation of atheroma formation and isassociated with the enhanced production of superoxide anion radical(O₂.-). Oxidation of LDL by O₂.- or other reactive species (e.g., .OH,ONO.O—, lipid peroxy radical, copper ion, and iron based proteins)reduces the affinity of LDL for uptake in cells via receptor mediatedendocytosis. Oxidatively modified LDLs are then rapidly taken up bymacrophages which subsequently transform into cells closely resemblingthe “foam cells” observed in early atherosclerotic lesions.

Oxidized lipoproteins can also promote vascular injury through theformation of lipid hydroperoxides within the LDL particle. This eventinitiates radical chain oxidation reactions of unsaturated LDL lipids,thus producing more oxidized LDL for macrophage incorporation.

The collective accumulation of foam cells engorged with oxidized LDLfrom these processes results in early “fatty streak” lesions, whicheventually progress to the more advanced complex lesions ofatherosclerosis leading to coronary disease.

As discussed generally by Jean Marx at page 320 of Science, Vol. 265(Jul. 15, 1994), each year about 330,000 patients in the United Statesundergo coronary and/or peripheral angioplasty, a procedure designed toopen up blood vessels, e.g., coronary arteries, clogged by dangerousatherosclerotic plaques (atherosclerosis) and thereby restore normalblood flow. For a majority of these patients, the operation works asintended. Nearly 33% of these patients (and maybe more by someaccounts), however, develop restenosis, wherein the treated arteriesbecome quickly clogged again. These patients are no better off, andsometimes worse off, than they were before angioplasty. Excessiveproliferation of smooth muscle cells (SMCs) in blood vessel wallscontributes to restenosis. Increased accumulation of oxidized LDL withinlesion SMCs might contribute to an atherogenic-related process likerestenosis. Zhou et al., “Association Between Prior CytomegalovirusInfection And The Risk Of Restenosis After Coronary Atherectomy,” Aug.29, 1996, New England Journal of Medicine, 335:624-630, and documentscited therein, all incorporated herein by reference. Accordingly,utility of the present invention with respect to atherosclerosis canapply to restenosis.

With regard to the inhibition by the inventive compounds ofcyclooxygenases (COX; prostaglandin endoperoxide synthase), it is knownthat cyclooxygenases are central enzymes in the production ofprostaglandins and other arachidonic acid metabolites (i.e.,eicosanoids) involved in many physiological processes. COX-1 is aconstitutive enzyme expressed in many tissues, including platelets,whereas COX-2, a second isoform of the enzyme, is inducible by variouscytokines, hormones and tumor promoters. COX 1 produces thromboxane A2,which is involved in platelet aggregation, which in turn is involved inthe progression of atherosclerosis. Its inhibition is the basis for theprophylactic effects on cardiovascular disease.

The activity of COX-1 and COX-2 is inhibited by aspirin and othernonsteroidal antiinflammatory drugs (NSAIDs), and the gastric sideeffects of NSAIDs are believed to be associated with the inhibition ofCOX-1. Moreover, it has been found that patients taking NSAIDs on aregular basis have a 40 to 50% lower risk of contracting colorectalcancer when compared to persons not being administered these type ofmedications; and COX-2 mRNA levels are markedly increased in 86% ofhuman colorectal adenocarcinomas.

One significant property of COX-2 expressing cell lines is the enhancedexpression of genes which participate in the modulation of apoptosis,i.e., programmed cell death. Several NSAIDs have been implicated inincreased cell death and the induction of apoptosis in chicken embryofibroblasts.

Cellular lipoxygenases are also involved in the oxidative modificationof LDL through the peroxidation of unsaturated lipids. The generation oflipid peroxy radicals contributes to the further radical chain oxidationof unsaturated LDL lipids, producing more oxidized LDL for macrophageincorporation.

It has been surprisingly found that the inventive compounds have utilityin the treatment of diseases associated with COX/LOX. In Example 28, COXwas inhibited by individual inventive compounds at concentrationssimilar to a known NSAID, indomethacin.

For COX inhibition, the inventive compounds are oligomers, where n is 2to 18. In a preferred embodiment, the inventive compounds are oligomerswhere n is 2 to 10, and more preferably, the inventive compounds areoligomers where n is 2 to 5.

Examples of compounds eliciting the inhibitory activity with respect toCOX/LOX cited above include dimers, trimers, tetramers and pentamers,discussed above.

Hence, given the significant inhibitory potency of the inventivecompounds on COX-2, coupled with the cytotoxic effects on a putativeCOX-2 expression colon cancer cell line, the inventive compounds possessapoptotic activity as inhibitors of the multistep progression leading tocarcinomas, as well as activity as members of the NSAID family ofmedications possessing a broad spectrum of prophylactic activities (see,e.g., Example 8, FIGS. 9D to 9H).

Further, prostaglandins, the penultimate products of the COX catalyzedconversion of arachidonic acid to prostaglandin H₂, are involved ininflammation, pain, fever, fetal development, labor and plateletaggregation. Therefore, the inventive compounds are efficacious for thesame conditions as NSAIDs, e.g., against cardiovascular disease, andstroke, etc. (indeed, the inhibition of platelet COX-1, which reducesthromboxane A₂ production, is the basis for the prophylactic effects ofaspirin on cardiovascular disease).

Inflammation is the response of living tissues to injury. It involves acomplex series of enzyme activation, mediator release, extravasation offluid, cell migration, tissue breakdown and repair. Inflammation isactivated by phospholipase A₂, which liberates arachidonic acid, thesubstrate for COX and LOX enzymes. COX converts arachidonic acid to theprostaglandin PGE₂, the major eicosanoid detected in inflammatoryconditions ranging from acute edema to chronic arthritis. Its inhibitionby NSAIDs is a mainstay for treatment.

Arthritis is one of the rheumatic diseases which encompass a wide rangeof diseases and pathological processes, most of which affect jointtissue. The basic structure affected by these diseases is the connectivetissue which includes synovial membranes, cartilage, bone, tendons,ligaments, and interstitial tissues. Temporary connective tissuesyndromes include sprains and strains, tendonitis, and tendon sheathabnormalities. The most serious forms of arthritis are rheumatoidarthritis, osteoarthritis, gout and systemic lupus erythematosus.

In addition to the rheumatic diseases, other diseases are characterizedby inflammation. Gingivitis and periodontitis follows a pathologicalpicture resembling rheumatoid arthritis. Inflammatory bowel diseaserefers to idiopathic chronic inflammatory conditions of the intestine,ulcerative colitis and Crohn's disease. Spondylitis refers to chronicinflammation of the joints of the spine. There is also a high incidenceof osteoarthritis associated with obesity.

Thus, the inventive compounds have utility in the treatment ofconditions involving inflammation, pain, fever, fetal development, laborand platelet aggregation.

The inhibition of COX by the inventive compounds would also inhibit theformation of postaglandins, e.g., PGD₂, PGE₂. Thus, the inventivecompounds have utility in the treatment of conditions associated withprostaglandin PGD₂, PGE₂

NO-Associated Utilities

Nitric oxide (NO) is known to inhibit platelet aggregation, monocyteadhesion and chemotaxis, and proliferation of vascular smooth muscletissue which are critically involved in the process of atherogenesis.Evidence supports the view that NO is reduced in atherosclerotic tissuesdue to its reaction with oxygen free radicals. The loss of NO due tothese reactions leads to increased platelet and inflammatory celladhesion to vessel walls to further impair NO mechanisms of relaxation.In this manner, the loss of NO promotes atherogenic processes, leadingto progressive disease states.

Hypertension is a leading cause of cardiovascular diseases, includingstroke, heart attack, heart failure, irregular heart beat and kidneyfailure. Hypertension is a condition where the pressure of blood withinthe blood vessels is higher than normal as it circulates through thebody. When the systolic pressure exceeds 150 mm Hg or the diastolicpressure exceeds 90 mm Hg for a sustained period of time, damage is doneto the body. For example, excessive systolic pressure can rupture bloodvessels anywhere. When it occurs within the brain, a stroke results. Itcan also cause thickening and narrowing of the blood vessels which canlead to atherosclerosis. Elevated blood pressure can also force theheart muscle to enlarge as it works harder to overcome the elevatedresting (diastolic) pressure when blood is expelled. This enlargementcan eventually produce irregular heart beats or heart failure.Hypertension is called the “silent killer” because it causes no symptomsand can only be detected when blood pressure is checked.

The regulation of blood pressure is a complex event where one mechanisminvolves the expression of constitutive Ca⁺²/calmodulin dependent formof nitric oxide synthase (NOS), abbreviated eNOS. NO produced by thisenzyme produces muscle relaxation in the vessel (dilation), which lowersthe blood pressure. When the normal level of NO produced by eNOS is notproduced, either because production is blocked by an inhibitor or inpathological states, such as atherosclerosis, the vascular muscles donot relax to the appropriate degree. The resulting vasoconstrictionincreases blood pressure and may be responsible for some forms ofhypertension.

Vascular endothelial cells contain eNOS. NO synthesized by eNOS diffusesin diverse directions, and when it reaches the underlying vascularsmooth muscle, NO binds to the heme group of guanylyl cyclase, causingan increase in cGMP. Increased cGMP causes a decrease in intracellularfree Ca⁺². Cyclic GMP may activate a protein kinase that phosphorylatesCa⁺² transporters, causing Ca⁺² to be sequestered in intracellularstructures in the muscle cells. Since muscle contraction requires Ca⁺²,the force of the contraction is reduced as the Ca⁺² concentrationdeclines. Muscle relaxation allows the vessel to dilate, which lowersthe blood pressure. Inhibition of eNOS therefore causes blood pressureto increase.

When the normal level of NO is not produced, either because productionis blocked by administration of an NOS inhibitor or possibly, inpathological states, such as atherosclerosis, the vascular muscles donot relax to the appropriate degree. The resulting vasoconstrictionincreases blood pressure and may be responsible for some forms ofhypertension. There is considerable interest in finding therapeutic waysto increase the activity of eNOS in hypertensive patients, but practicaltherapies have not been reported. Pharmacological agents capable ofreleasing NO, such as nitroglycerin or isosorbide dinitrate, remainmainstays of vasorelaxant therapy.

Although the inventive compounds inhibit the oxidation of LDL, the morecomprehensive effects of these compounds is their multidimensionaleffects on atherosclerosis via NO. NO modulation by the inventivecompounds brings about a collage of beneficial effects, including themodulation of hypertension, lowering NO affected hypercholesterolemia,inhibiting platelet aggregation and monocyte adhesion, all of which areinvolved with the progression of atherosclerosis.

The role of NO in the immune system is different from its function inblood vessels. Macrophages contain a form of NOS that is inducible,rather than constitutive, referred to as iNOS. Transcription of the iNOSgene is controlled both positively and negatively by a number ofbiological response modifiers called cytokines. The most importantinducers are gamma-interferon, tumor necrosis factor, interleukin-1,interleukin-2 and lipopolysaccharide (LPS), which is a component of thecell walls of gram negative bacteria. Stimulated macrophages produceenough NO to inhibit ribonuclease reductase, the enzyme that convertsribonucleotides to the deoxyribonucleotides necessary for DNA synthesis.Inhibition of DNA synthesis may be an important way in which macrophagesand other tissues possessing iNOS can inhibit the growth of rapidlydividing tumor cells or infectious bacteria.

With regard to the effects of NO and infectious bacteria, microorganismsplay a significant role in infectious processes which reflect bodycontact and injury, habits, profession, environment of the individual,as well as food borne diseases brought about by improper storage,handling and contamination.

The inventive compounds, combinations thereof and compositionscontaining the same are useful in the treatment of conditions associatedwith modulating NO concentrations.

Example 9 described the antioxidant activity (as inhibitors of freeradicals) of the inventive compounds. Given that NO is a free radicaland that the inventive compounds are strong antioxidants, it wassuspected that the administration of the inventive compounds toexperimental in vitro and in vivo models would have caused a reductionin NO levels. Any reduction in NO would have resulted in a hypertensive,rather than a hypotensive effect. Contrary to expectations, theinventive compounds elicited increases in NO from in vitro experimentsand produced a hypotensive effect from in vivo studies (Examples 31 and32). These results were unanticipated and completely unexpected.

Example 27 describes an erythmia (facial flush) shortly after drinking asolution containing the inventive compounds and glucose, thus implying avasodilation effect.

Example 31 describes the hypotensive effects elicited by the inventivecompounds in an in vivo animal model, demonstrating the efficacy of theinventive compounds in the treatment of hypertension. In this example,the inventive compounds, combinations thereof and compositionscomprising the same comprise oligomers wherein n is 2 to 18, andpreferably, n is 2 to 10.

Example 32 describes the modulation of NO production by the inventivecompounds in an in vitro model. In this example, the inventivecompounds, combinations thereof and compositions comprising the samecomprise oligomers wherein n is 2 to 18, and preferably n is 2 to 10.

Further, Example 35 provides evidence for the formation of Cu⁺²—, Fe⁺²—and Fe⁺³— oligomer complexes detected by MALDI/TOF/MS. These resultsindicate that the inventive compounds can complex with copper and/oriron ions to minimize their effects on LDL oxidation.

Moreover, the inventive compounds have useful anti-microbial activitiesfor the treatment of infections and for the prevention of food spoilage.Examples 22 and 30 describe the antimicrobial activity of the inventivecompounds against several representative microbiota having clinical andfood significance, as outlined below. CLINICAL/FOOD MICROORGANISM TYPERELEVANCE Helicobacter pylori gram negative gastritis, ulcers, gastriccancer Bacillus species gram positive food poisoning, wound infections,bovine mastitis, septicemia Salmonella species gram negative foodpoisoning, diarrhea Staphylococcus aureus gram positive boils,carbuncles, wound infection, septicemia, breast abscesses Escherichiacoli gram negative infant diarrhea, urinary tract infection Pseudomonasspecies gram negative urinary tract infections, wound infections,“swimmer's ear” Saccharomyces cervisea yeast food spoilage Acetobacterpasteurianus gram negative food spoilage

Example 33 describes the effects of the inventive compounds onmacrophage NO production. In this example, the results demonstrate thatthe inventive compounds induce monocyte/macrophage NO production, bothindependent and dependent of stimulation by lipopolysaccharide (LPS) orcytokines. Macrophages producing NO can inhibit the growth of infectiousbacteria.

Compounds of the invention eliciting antimicrobial activity areoligomers, where n is 2 to 18, and preferably, are oligomers where n is2, 4, 5, 6, 8 and 10.

Examples of compounds eliciting the antimicrobial activity with respectto NO cited above include dimers, tetramers, pentamers, hexamers,octamers and decamers, discussed above.

Anti-Cancer Utilities

Cancers are classified into three groups: carcinomas, sarcomas andlymphomas. A carcinoma is a malignancy that arises in the skin, liningsof various organs, glands and tissues. A sarcoma is a malignancy thatarises in the bone, muscle or connective tissue. The third groupcomprises leukemias and lymphomas because both develop within the bloodcell forming organs. The major types of cancer are prostate, breast,lung, colorectal, bladder, non-Hodgkin's lymphoma, uterine, melanoma ofthe skin, kidney, leukemia, ovarian and pancreatic.

The development of cancer results from alterations to the DNA of cellswhich is brought about by many factors such as inheritable geneticfactors, ionizing radiation, pollutants, radon, and free radical damageto the DNA. Cells carrying mutations produce a defect in the orderedprocess of cell division. These cells fail to undergo apoptosis(programmed cell death) and continue to divide which either marks thebeginnings of a malignant tumor or allows more mutations to occur overtime to result in a malignancy.

There are three major features common to the many different cancers.These are (1) the ability to proliferate indefinitely; (2) invasion ofthe tumor into the surrounding tissue; and (3) the process ofmetastasis.

Certain types of cancer metastasize in characteristic ways. For example,cancers of the thyroid gland, lung, breast, kidney and prostate glandfrequently metastasize to the bones. Lung cancer commonly spreads to thebrain and adrenal glands and colorectal cancer often metastasizes to theliver. Leukemia is considered to be a generalized disease at the onset,where it is found in the bone marrow throughout the body.

It has been surprisingly found that the inventive compounds are usefulin the treatment of a variety of cancers discussed above. Examples 6, 7,8 and 15 describe the inventive compounds which elicit anti-canceractivity against human HeLa (cervical), prostate, breast, renal, T-cellleukemia and colon cancer cell lines. Example 12 (FIG. 20) illustratesthe dose response effects on HeLa and SKBR-3 breast cancer cell linestreated with oligomeric (dimers-dodecamers) procyanidins, which weresubstantially purified by HPLC. Cytotoxicity against these cancer celllines were dependent upon pentamer through dodecamer procyanidins, withthe lower oligomers showing no effect.

While not wishing to be bound by any theory, there appeared to be aminimum structural motif that accounts for the effects described above.Example 37 also shows the same cytotoxic effects of the higher oligomers(pentamer-decamer) against a feline lymphoblastoid cancer cell line.Cytotoxicity was also observed with higher oligomers (FIGS. 58 to 61)against normal canine and feline cell lines.

In Example 8 (FIGS. 9D-H), the inventive compounds were shown to elicitcytotoxicity against a putative COX-2 expressing human colon cancer cellline (HCT 116).

Example 9 describes the antioxidant activity by the inventive compounds.The compounds of the invention inhibit DNA strand breaks, DNA-proteincross-links and free radical oxidation of nucleotides to reduce and/orprevent the occurrence of mutations.

Example 10 describes the inventive compounds as topoisomerase IIinhibitors, which is a target for chemotherapeutic agents, such asdoxorubicin.

Example 21 describes the in vivo effects of a substantially purepentamer which elicited anti-tumor activity against a human breastcancer cell line (MDA-MB-231/LCC6) in a nude mouse model (average weightof a mouse is approximately 25 g). Repeat in vivo experiments with thepentamer at higher dosages (5 mg) have not entirely been successful, dueto unexpected animal toxicity. It is currently believed that thistoxicity may be related to the vasodilation effects of the inventivecompounds.

Example 33 describes the effects of the inventive compounds onmacrophage NO production. Macrophages which produce NO can inhibit thegrowth of rapidly dividing tumor cells.

Still further, the invention includes the use of the inventive compoundsto induce the inhibition of cellular proliferation by apoptosis.

For anti-cancer activity, the inventive compounds are oligomers, where nis 2 to 18, e.g., 3 to 18, such as 3 to 12, and preferably, n is 5 to12, and most preferably n is 5.

Compounds which elicit the inhibitory activity with respect to cancercited above include pentamers to dodecamers, discussed above.

Formulations and Methods

Therefore, collectively, the inventive compounds, combinations thereofand compositions comprising the same have exhibited a wide array ofactivities against several aspects of atherosclerosis, cardiovasculardisease, cancer, blood pressure modulation and/or hypertension,inflammatory disease, infectious agents and food spoilage.

Hence, the compounds of the invention, combinations thereof andcompositions containing the same are COX inhibitors which affectplatelet aggregation by inhibiting thromboxane A₂ formation, thusreducing the risk for thrombosis. Further, the inhibition of COX leadsto decreased platelet and inflammatory cell adhesion to vessel walls toallow for improved NO mechanisms of relaxation. These results, coupledwith the inhibition of COX at concentrations similar to a known NSAID,indomethacin, indicates antithrombotic efficacy.

Moreover, the compounds of the invention, combinations thereof andcompositions containing the same are antioxidants which suppress theoxidation of LDL by reducing the levels of superoxide radical anion andlipoxygenase mediated lipid peroxy radicals. The inhibition of LDLoxidation at this stage slows macrophage activation and retards foamcell formation to interrupt further progression of atherosclerosis. Theinhibition of LDL oxidation can also slow the progression of restenosis.Thus, compounds of the invention or combinations thereof or compositionscontaining compounds of the invention or combinations thereof can beused for prevention and/or treatment of atherosclerosis and/orrestenosis. And thus, the inventive compounds can be administered beforeor after angioplasty or similar procedures to prevent or treatrestenosis in patients susceptible thereto.

For treatment or prevention of restenosis and/or atherosclerosis, aninventive compound or compounds or a composition comprising an inventivecompound or compounds, alone or with other treatment, may beadministered as desired by the skilled medical practitioner, from thisdisclosure and knowledge in the art, e.g., at the first signs orsymptoms of restenosis and/or atherosclerosis, immediately prior to,concomitant with or after angioplasty, or as soon thereafter as desiredby the skilled medical practitioner, without any undue experimentationrequired; and the administration of the inventive compound or compoundsor a composition thereof, alone or with other treatment, may becontinued as a regimen, e.g., monthly, bimonthly, biannually, annually,or in some other regimen, by the skilled medical practitioner for suchtime as is necessary, without any undue experimentation required.

Further, the compounds of the invention, combinations thereof andcompositions comprising the same have been shown to produce ahypotensive effect in vivo and induce NO in vitro. These results havepractical application in the treatment of hypertension and in clinicalsituations involving hypercholesterolemia, where NO levels are markedlyreduced.

Formulations of the inventive compounds, combinations thereof andcompositions comprising the same can be prepared with standardtechniques well known to those skilled in the pharmaceutical, foodscience, medical and veterinary arts, in the form of a liquid,suspension, tablet, capsule, injectable solution or suppository, forimmediate or slow-release of the active compounds.

The carrier may also be a polymeric delayed release system. Syntheticpolymers are particularly useful in the formulation of a compositionhaving controlled release. An early example of this was thepolymerization of methyl methacrylate into spheres having diameters lessthan one micron to form so-called nano particles, reported by Kreuter,J., Microcapsules and Nanoparticles in Medicine and Pharmacology, M.Donbrow (Ed). CRC Press, p. 125-148.

A frequent choice of a carrier for pharmaceuticals and more recently forantigens is poly (d,l-lactide-co-glycolide) (PLGA). This is abiodegradable polyester that has a long history of medical use inerodible sutures, bone plates and other temporary prostheses where ithas not exhibited any toxicity. A wide variety of pharmaceuticals havebeen formulated into PLGA microcapsules. A body of data has accumulatedon the adaption of PLGA for controlled, for example, as reviewed byEldridge, J. H., et al. Current Topics in Microbiology and Immunology.1989, 146:59-66. The entrapment in PLGA microspheres of 1 to 10 micronsin diameter can have an effect when administered orally. The PLGAmicroencapsulation process uses a phase separation of a water-in-oilemulsion. The inventive compound or compounds is or are prepared as anaqueous solution and the PLGA is dissolved in a suitable organicsolvents such as methylene chloride and ethyl acetate. These twoimmiscible solutions are co-emulsified by high-speed stirring. Anon-solvent for the polymer is then added, causing precipitation of thepolymer around the aqueous droplets to form embryonic microcapsules. Themicrocapsules are collected, and stabilized with one of an assortment ofagents (polyvinyl alcohol (PVA), gelatin, alginates, methyl cellulose)and the solvent removed by either drying in vacuo or solvent extraction.

Additionally, with regard to the preparation of slow-releaseformulations, reference is made to U.S. Pat. Nos. 5,024,843, 5,091,190,5,082,668, 4,612,008 and 4,327,725, hereby incorporated herein byreference.

Additionally, selective processing coupled with the identification ofcocoa genotypes of interest could be used to prepareStandard-of-Identity (SOI) and non-SOI chocolate products as vehicles todeliver the active compounds to a patient in need of treatment for thedisease conditions described above, as well as a means for the deliveryof conserved levels of the inventive compounds.

In this regard, reference is made to copending U.S. application Ser. No.08/709,406, filed Sep. 6, 1996 (now U.S. Pat. No. 6,015,913), herebyincorporated herein by reference. U.S. Ser. No. 08/709,406 (now U.S.Pat. No. 6,015,913) relates to a method of producing cocoa butter and/orcocoa solids having conserved levels of polyphenols from cocoa beansusing a unique combination of processing steps which does not requireseparate bean roasting or liquor milling equipment, allowing for theoption of processing cocoa beans without exposure to severe thermaltreatment for extended periods of time and/or the use of solventextraction of fat. The benefit of this process lies in the enhancedconservation of polyphenols in contrast to that found in traditionalcocoa processing, such that the ratio of the initial amount ofpolyphenol found in the unprocessed bean to that obtainable afterprocessing is less than or equal to 2.

Compositions of the invention include one or more of the above notedcompounds in a formulation having a pharmaceutically acceptable carrieror excipient, the inventive compounds having anti-cancer, anti-tumor orantineoplastic activities, antioxidant activity, inhibit DNAtopoisomeriase II enzyme, inhibit oxidative damage to DNA, inducemonocyte/macrophage NO production, have antimicrobial, cyclo-oxygenaseand/or lipoxygenase, NO or NO-synthase, apoptosis, platelet aggregationand blood or in vivo glucose modulating activities, and have efficacy asnon-steroidal antiinflammatory agents.

Another embodiment of the invention includes compositions comprising theinventive compounds or combinations thereof, as well as at least oneadditional antineoplastic, blood pressure reducing, antiinflammatory,antimicrobial, antioxidant and hematopoiesis agents, in addition to apharmaceutically acceptable carrier or excipient.

Such compositions can be administered to a subject or patient in need ofsuch administration in dosages and by techniques well known to thoseskilled in the medical, nutritional or veterinary arts taking intoconsideration the data herein, and such factors as the age, sex, weight,genetics and condition of the particular subject or patient, and theroute of administration, relative concentration of particular oligomers,and toxicity (e.g., LD₅₀).

The compositions can be co-administered or sequentially administeredwith other antineoplastic, anti-tumor or anti-cancer agents,antioxidants, DNA topoisomerase II enzyme inhibiting agents, inhibitorsof oxidatively damaged DNA or cyclo-oxygenase and/or lipoxygenase,apoptosis, platelet aggregation, blood or in vivo glucose or NO orNO-synthase modulating agents, non-steroidal antiinflammatory agentsand/or with agents which reduce or alleviate ill effects ofantineoplastic, anti-tumor, anti-cancer agents, antioxidants, DNAtopoisomerase II enzyme inhibiting agents, inhibitors of oxidativelydamaged DNA, cyclo-oxygenase and/or lipoxygenase, apoptosis, plateletaggregation, blood or in vivo glucose or NO or NO-synthase modulatingand/or non-steroidal antiinflammatory agents; again, taking intoconsideration such factors as the age, sex, weight, genetics andcondition of the particular subject or patient, and, the route ofadministration.

Examples of compositions of the invention for human or veterinary useinclude edible compositions for oral administration, such solid orliquid formulations, for instance, capsules, tablets, pills and thelike, as well as chewable solid or beverage formulations, to which thepresent invention may be well-suited since it is from an edible source(e.g., cocoa or chocolate flavored solid or liquid compositions); liquidpreparations for orifice, e.g., oral, nasal, anal, vaginal etc.,administration such as suspensions, syrups or elixirs (including cocoaor chocolate flavored compositions); and, preparations for parental,subcutaneous, intradermal, intramuscular or intravenous administration(e.g., injectable administration) such as sterile suspensions oremulsions. However, the active ingredient in the compositions maycomplex with proteins such that when administered into the bloodstream,clotting may occur due to precipitation of blood proteins; and, theskilled artisan should take this into account. In such compositions theactive cocoa extract may be in admixture with a suitable carrier,diluent, or excipient such as sterile water, physiological saline,glucose, DMSO, ethanol, or the like. The active cocoa extract of theinvention can be provided in lyophilized form for reconstituting, forinstance, in isotonic aqueous, saline, glucose or DMSO buffer. Incertain saline solutions, some precipitation has been observed; and,this observation may be employed as a means to isolate inventivecompounds, e.g., by a “salting out” procedure.

Example 38 describes the preparation of the inventive compounds in atablet formulation for application in the pharmaceutical, supplement andfood areas. Further, Example 39 describes the preparation of theinventive compounds in capsule formulations for similar applications.Still further, Example 40 describes the formulation of Standard ofIdentity (SOI) and non-SOI chocolates containing the compounds of theinvention or cocoa solids obtained from methods described in copendingU.S. application Ser. No. 08/709,406 (now U.S. Pat. No. 6,015,913),hereby incorporated herein by reference.

Kits

Further, the invention also comprehends a kit wherein the active cocoaextract is provided. The kit can include a separate container containinga suitable carrier, diluent or excipient. The kit can also include anadditional anti-cancer, anti-tumor or antineoplastic agent, antioxidant,DNA topoisomerase II enzyme inhibitor or an inhibitor of oxidative DNAdamage or antimicrobial, or cyclo-oxygenase and/or lipoxygenase, NO orNO-synthase non-steroidal antiinflammatory, apoptosis and plateletaggregation modulating or blood or in vivo glucose modulating agentand/or an agent which reduces or alleviates ill effects ofantineoplastic, anti-tumor or anti-cancer agents, antioxidant, DNAtopoisomerase II enzyme inhibitor or antimicrobial, or cyclo-oxygenaseand/or lipoxygenase, NO or NO-synthase, apoptosis, platelet aggregationand blood or in vivo glucose modulating and/or non-steroidalantiinflammatory agents for co- or sequential-administration. Theadditional agent(s) can be provided in separate container(s) or inadmixture with the active cocoa extract. Additionally, the kit caninclude instructions for mixing or combining ingredients and/oradministration.

Identification of Genes

A further embodiment of the invention comprehends the modulation ofgenes expressed as a result of intimate cellular contact by theinventive compounds or a combination of compounds. As such, the presentinvention comprehends methods for the identification of genes induced orrepressed by the inventive compounds or a combination of compounds whichare associated with several diseases, including but not limited toatherosclerosis, hypertension, cancer, cardiovascular disease, andinflammation. Specifically, genes which are differentially expressed inthese disease states, relative to their expression in “normal”nondisease states are identified and described before and after contactby the inventive compounds or a combination of compounds.

As mentioned in the previous discussion, these diseases and diseasestates are based in part on free radical interactions with a diversityof biomolecules. A central theme in these diseases is that many of thefree radical reactions involve reactive oxygen species, which in turninduce physiological conditions involved in disease progression. Forinstance, reactive oxygen species have been implicated in the regulationof transcription factors such as nuclear factor (NF)-κB. The targetgenes for NF-κB comprise a list of genes linked to coordinatedinflammatory response. These include genes encoding tumor necrosisfactor (TNF)-α, interleukin (IL)-1, IL-6, IL-8, inducible NOS, MajorHistocompatabilty Complex (MHC) class I antigens, and others. Also,genes that modulate the activity of transcription factors may in turn beinduced by oxidative stress. Oxidative stress is the imbalance betweenradical scavenging and radical generating systems. Several knownexamples (Winyard and Blake, 1997) of these conditions include gadd153(a gene induced by growth arrest and DNA damage), the product of whichhas been shown to bind NF-IL6 and form a heterodimer that cannot bind toDNA. NF-IL6 upregulates the expression of several genes, including thoseencoding interleukins 6 and 8. Another example of oxidative stressinducible genes are gadd45 which regulates the effects of thetranscription factor p53 in growth arrest. p53 codes for the p53 proteinwhich can halt cell division and induce abnormal cells (e.g. cancer) toundergo apoptosis.

Given the full panoply of unexpected, nonobvious and novel utilities forthe inventive compounds or combination of compounds for utility in adiverse array of diseases based in part by free radical mechanisms, theinvention further comprehends strategies to determine the temporaleffects on gene(s) or gene product(s) expression by the inventivecompounds in animal in vitro and/or in vivo models of specific diseaseor disease states using gene expression assays. These assays include,but are not limited to Differential Display, sequencing of cDNAlibraries, Serial Analysis of Gene Expression (SAGE), expressionmonitoring by hybridization to high density oligonucleotide arrays andvarious reverse transcriptase-polymerization chain reaction (RT-PCR)based protocols or their combinations (Lockhart et al., 1996).

The comprehensive physiological effects of the inventive compounds orcombination of compounds embodied in the invention, coupled to a geneticevaluation process permits the discovery of genes and gene products,whether known or novel, induced or repressed. For instance, theinvention comprehends the in vitro and in vivo induction and/orrepression of cytokines (e.g. IL-1, IL-2, IL-6, IL-8, IL-12, and TNF-α)in lymphocytes using RT-PCR. Similarly, the invention comprehends theapplication of Differential Display to ascertain the induction and/orrepression of select genes; for the cardiovascular area (e.g. superoxidedismutase, heme oxidase, COX 1 and 2, and other oxidant defense genes)under stimulated and/or oxidant stimulated conditions (e.g. TNF-α orH₂O₂) conditions. For the cancer area, the invention comprehends theapplication of Differential Display to ascertain the induction and/orrepression of genes or gene products such as CuZn-superoxide dismutase,Mn-superoxide dismutase, catalase, etc., in control and oxidant stressedcells.

The following non-limiting Examples are given by way of illustrationonly and are not to be considered a limitation of this invention, manyapparent variations of which are possible without departing from thespirit or scope thereof.

EXAMPLES Example 1 Cocoa Source and Method of Preparation

Several Theobroma cacao genotypes which represent the three recognizedhorticultural races of cocoa (Enriquez, 1967; Engels, 1981) wereobtained from the three major cocoa producing origins of the world. Alist of those genotypes used in this study are shown in Table 1.Harvested cocoa pods were opened and the beans with pulp were removedfor freeze drying. The pulp was manually removed from the freeze driedmass and the beans were subjected to analysis as follows. Theunfermented, freeze dried cocoa beans were first manually dehulled, andground to a fine powdery mass with a TEKMAR Mill. The resultant mass wasthen defatted overnight by Soxhlet extraction using redistilled hexaneas the solvent. Residual solvent was removed from the defatted mass byvacuum at ambient temperature. TABLE 1 Description of Theobroma cacaoSource Material GENOTYPE ORIGIN HORTICULTURAL RACE UIT-1 MalaysiaTrinitario Unknown West Africa Forastero ICS-100 Brazil Trinitario(Nicaraguan Criollo ancestor) ICS-39 Brazil Trinitario (NicaraguanCriollo ancestor) UF-613 Brazil Trinitario EEG-48 Brazil Forastero UF-12Brazil Trinitario NA-33 Brazil Forastero

Example 2 Procyanidin Extraction Procedures

A. Method 1

Procyanidins were extracted from the defatted, unfermented, freeze driedcocoa beans of Example 1 using a modification of the method described byJalal and Collin (1977). Procyanidins were extracted from 50 grambatches of the defatted cocoa mass with 2×400 mL 70% acetone/deionizedwater followed by 400 mL 70% methanol/deionized water. The extracts werepooled and the solvents removed by evaporation at 45° C. with a rotaryevaporator held under partial vacuum. The resultant aqueous phase wasdiluted to 1 L with deionized water and extracted 2× with 400 mL CHCl₃.The solvent phase was discarded. The aqueous phase was then extracted 4×with 500 mL ethyl acetate. Any resultant emulsions were broken bycentrifugation on a Sorvall RC 28S centrifuge operated at 2,000×g for 30min. at 10° C. To the combined ethyl acetate extracts, 100-200 mLdeionized water was added. The solvent was removed by evaporation at 45°C. with a rotary evaporator held under partial vacuum. The resultantaqueous phase was frozen in liquid N₂ followed by freeze drying on aLABCONCO Freeze Dry System. The yields of crude procyanidins that wereobtained from the different cocoa genotypes are listed in Table 2. TABLE2 Crude Procyanidin Yields GENOTYPE ORIGIN YIELDS (g) UIT-1 Malaysia3.81 Unknown West Africa 2.55 ICS-100 Brazil 3.42 ICS-39 Brazil 3.45UF-613 Brazil 2.98 EEG-48 Brazil 3.15 UF-12 Brazil 1.21 NA-33 Brazil2.23B. Method 2

Alternatively, procyanidins are extracted from defatted, unfermented,freeze dried cocoa beans of Example 1 with 70% aqueous acetone. Tengrams of defatted material was slurried with 100 mL solvent for 5-10min. The slurry was centrifuged for 15 min. at 4° C. at 3000×g and thesupernatant passed through glass wool. The filtrate was subjected todistillation under partial vacuum and the resultant aqueous phase frozenin liquid N₂, followed by freeze drying on a LABCONCO Freeze Dry System.The yields of crude procyanidins ranged from 15-20%.

Without wishing to be bound by any particular theory, it is believedthat the differences in crude yields reflected variations encounteredwith different genotypes, geographical origin, horticultural race, andmethod of preparation.

Example 3 Partial Purification of Cocoa Procyanidins

Procyanidins obtained from Example 2 were partially purified by liquidchromatography on Sephadex LH-20 (28×2.5 cm). Separations were aided bya step gradient from deionized water into methanol. The initial gradientcomposition started with 15% methanol in deionized water which wasfollowed step wise every 30 min. with 25% methanol in deionized water,35% methanol in deionized water, 70% methanol in deionized water, andfinally 100% methanol. The effluent following the elution of thexanthine alkaloids (caffeine and theobromine) was collected as a singlefraction. The fraction yielded a xanthine alkaloid free subfractionwhich was submitted to further subfractionation to yield fivesubfractions designated MM2A through MM2E. The solvent was removed fromeach subfraction by evaporation at 4°5C with a rotary evaporator heldunder partial vacuum. The resultant aqueous phase was frozen in liquidN₂ and freeze dried overnight on a LABCONCO Freeze Dry System. Arepresentative gel permeation chromatogram showing the fractionation isshown in FIG. 1. Approximately, 100 mg of material was subfractionatedin this manner.

Chromatographic Conditions: Column; 28×2.5 cm Sephadex LH-20, MobilePhase: Methanol/Water Step Gradient, 15:85, 25:75, 35:65, 70:30, 100:0Stepped at ½ Hour Intervals, Flow Rate; 1.5 mL/min, Detector; UV atλ₁=254 nm and λ₂=365 nm, Chart Speed: 0.5 mm/min, Column Load; 120 mg.

B. Semi-preparative High Performance Liquid Chromatography (HPLC)

Method 1. Reverse Phase Separation

Procyanidins obtained from Example 2 and/or 3A were partially purifiedby semi-preparative HPLC. A Hewlett Packard 1050 HPLC System equippedwith a variable wavelength detector, Rheodyne 7010 injection valve with1 mL injection loop was assembled with a Pharmacia FRAC-100 FractionCollector. Separations were effected on a Phenomenex Ultracarb™ 10μ ODScolumn (250×22.5 mm) connected with a Phenomenex 10μ ODS Ultracarb™(60×10 mm) guard column. The mobile phase composition was A=water;B=methanol used under the following linear gradient conditions: [Time, %A]; (0,85), (60,50), (90,0), and (110,0) at a flow rate of 5 mL/min.Compounds were detected by UV at 254 nm.

A representative Semi-preparative HPLC trace is shown in FIG. 15N forthe separation of procyanidins present in fraction D+E. Individual peaksor select chromatographic regions were collected on timed intervals ormanually by fraction collection for further purification and subsequentevaluation. Injection loads ranged from 25-100 mg of material.

Method 2. Normal Phase Separation

Procyanidin extracts obtained from Examples 2 and/or 3A were partiallypurified by semi-preparative HPLC. A Hewlett Packard 1050 HPLC system,Millipore-Waters Model 480 LC detector set at 254 nm was assembled witha Pharmacia Frac-100 Fraction Collector set in peak mode. Separationswere effected on a Supelco 5 μm Supelcosil LC-Si column (250×10 mm)connected with a Supelco 5 μm Supelguard LC-Si guard column (20×4.6 mm).Procyanidins were eluted by a linear gradient under the followingconditions: (Time, % A, % B); (0,82,14), (30, 67.6, 28.4), (60, 46, 50),(65, 10, 86), (70, 10, 86) followed by a 10 min. re-equilibration.Mobile phase composition was A=dichloromethane; B=methanol; and C=aceticacid: water (1:1). A flow rate of 3 mL/min was used. Components weredetected by UV at 254 nm, and recorded on a Kipp & Zonan BD41 recorder.Injection volumes ranged from 100-250 μL of 10 mg of procyanidinextracts dissolved in 0.25 mL 70% aqueous acetone. A representativesemi-preparative HPLC trace is shown in FIG. 15 O. Individual peaks orselect chromatographic regions were collected on timed intervals ormanually by fraction collection for further purification and subsequentevaluation.

HPLC Conditions:

-   -   250×10 mm Supelco Supelcosil LC-Si (5 μm) Semipreparative Column    -   20×4.6 mm Supelco Supelcosil LC-Si (5 μm) Guard Column    -   Detector: Waters LC    -   Spectrophotometer Model 480@254 nm    -   Flow rate: 3 mL/min,    -   Column Temperature: ambient,    -   Injection: 250 lL of 70% aqueous

acetone extract. Gradient: Acetic Acid:H₂0 Time (min) CH₂Cl₂ Methanol(1:1) 0 82 14 4 30 67.6 28.4 4 60 46 50 4 65 10 86 4 70 10 86 4

The fractions obtained were as follows: FRACTION TYPE 1 dimers 2 trimers3 tetramers 4 pentamers 5 hexamers 6 heptamers 7 octamers 8 nonamers 9decamers 10 undecamers 11 dodecamers 12 higher oligomers

Example 4 Analytical HPLC Analysis of Procyanidin Extracts

Method 1. Reverse Phase Separation

Procyanidin extracts obtained from Example 3 were filtered through a0.45μ filter and analyzed by a Hewlett Packard 1090 ternary HPLC systemequipped with a Diode Array detector and a HP model 1046A ProgrammableFluorescence Detector. Separations were effected at 45° C. on aHewlett-Packard 51 Hypersil ODS column (200×2.1 mm). The flavanols andprocyanidins were eluted with a linear gradient of 60% B into A followedby a column wash with B at a flow rate of 0.3 mL/min. The model phasecomposition was B=0.5% acetic acid in methanol and A=0.5% acetic acid innanopure water. Acetic acid levels in A and B mobile phases can beincreased to 2%. Components were detected by fluorescence, whereλ_(ex)=276 nm and λ_(ex)=316 nm and by UV at 280 nm. Concentrations of(+)-catechin and (−)-epicatechin were determined relative to referencestandard solutions. Procyanidin levels were estimated by using theresponse factor for (−)-epicatechin. A representative HPLC chromatogramshowing the separation of the various components is shown in FIG. 2A forone cocoa genotype. Similar HPLC profiles were obtained from the othercocoa genotypes.

HPLC Conditions:

-   -   Column: 200×2.1 mm Hewlett Packard Hypersil ODS (5μ)    -   Guard column: 20×2.1 mm Hewlett Packard Hypersil ODS (5μ)    -   Detectors:        -   Diode Array@280 nm        -   Fluorescence λex=276 nm;        -   λm=316 nm.    -   Flow rate: 0.3 mL/min.

Column Temperature: 45° C. Gradient: 0.5% Acetic Acid in 0.5% Aceticacid Time (min) nanopure water in methanol 0 100 0 50 40 60 60 0 100

Method 2. Normal Phase Separation

Procyanidin extracts obtained from Examples 2 and/or 3 were filteredthrough a 0.45μ filter and analyzed by a Hewlett Packard 1090 Series IIHPLC system equipped with a HP model 1046A Programmable Fluorescencedetector and Diode Array detector. Separations were effected at 37° C.on a 5 μ Phenomenex Lichrosphere® Silica 100 column (250×3.2 mm)connected to a Supelco Supelguard LC-Si 5μ guard column (20×4.6 mm).Procyanidins were eluted by linear gradient under the followingconditions: (Time, % A, % B); (0, 82, 14), (30, 67.6, 28.4), (60, 46,50), (65, 10, 86), (70, 10, 86) followed by an 8 min. re-equilibration.Mobile phase composition was A=dichloromethane, B=methanol, and C=aceticacid: water at a volume ratio of 1:1. A flow rate of 0.5 mL/min. wasused. Components were detected by fluorescence, where λ_(ex)=276 nm andλ_(em)=316 nm or by UV at 280 nm. A representative HPLC chromatogramshowing the separation of the various procyanidins is shown in FIG. 2Bfor one genotype. Similar HPLC profiles were obtained from other cocoagenotypes.

HPLC Conditions:

-   -   250×3.2 mm Phenomenex Lichrosphere® Silica 100 column (51)        20×4.6 mm Supelco Supelguard LC-Si (5μ) guard column    -   Detectors:        -   Photodiode Array@280 nm        -   Fluorescence λ_(ex)=276 nm;        -   λ_(em)=316 nm.    -   Flow rate: 0.5 mL/min.

Column Temperature: 37° C. Gradient: Acetic Acid/Water Time (min.)CH₂—Cl₂ Methanol (1:1) 0 82 14 4 30 67.6 28.4 4 60 46 50 4 65 10 86 4 7010 86 4

Example 5 Identification of Procyanidins

Procyanidins were purified by liquid chromatography on Sephadex LH-20(28×2.5 cm) columns followed by semi-preparative HPLC using a 10μBondapak C18 (100×8 mm) column or by semi-preparative HPLC using a 5μSupelcosil LC-Si (250×10 mm) column.

Partially purified isolates were analyzed by Fast Atom Bombardment—MassSpectrometry (FAB-MS) on a VG ZAB-T high resolution MS system using aLiquid Secondary Ion Mass Spectrometry (LSIMS) technique in positive andnegative ion modes. A cesium ion gun was used as the ionizing source at30 kV and a “Magic Bullet Matrix” (1:1 dithiothreitol/dithioerythritol)was used as the proton donor.

Analytical investigations of these fractions by LSIMS revealed thepresence of a number of flavan-3-ol oligomers as shown in Table 3. TABLE3 LSIMS (Positive Ion) Data from Cocoa Procyanidin Fractions (M + 1)⁺(M + Na)⁺ Oligomer m/z m/z Mol. Wt. Monomers (catechins)  291  313  290Dimer(s) 577/579 599/601 576/578 Trimer(s) 865/867 887/889 864/866Tetramer(s) 1155 1177 1154 Pentamer(s) 1443 1465 1442 Hexamer(s) 17311753 1730 Heptamer(s) — 2041 2018 Octamer(s) — 2329 2306 Nonamer(s) —2617 2594 Decamer(s) — 2905 2882 Undecamer(s) — — 3170 Dodecamer(s) — —3458

The major mass fragment ions were consistent with work previouslyreported for both positive and negative ion FAB-MS analysis ofprocyanidins (Self et al., 1986 and Porter et al., 1991). The ioncorresponding to m/z 577 (M+H)⁺ and its sodium adduct at m/z 599 (M+Na)⁺suggested the presence of doubly linked procyanidin dimers in theisolates. It was interesting to note that the higher oligomers were morelikely to form sodium adducts (M+Na)⁺ than their protonated molecularions (M+H)⁺. The procyanidin isomers B-2, B-5 and C-1 were tentativelyidentified based on the work reported by Revilla et al. (1991), Self etal. (1986) and Porter et al. (1991). Procyanidins up to both the octamerand decamer were verified by FAB-MS in the partially purified fractions.Additionally, evidence for procyanidins up to the dodecamer wereobserved from normal phase HPLC analysis (see FIG. 2B). Table 4 liststhe relative concentrations of the procyanidins found in xanthinealkaloid free isolates based on reverse phase HPLC analysis. Table 5lists the relative concentrations of the procyanidins based on normalphase HPLC analysis. TABLE 4 Relative Concentrations of Procyanidins inthe Xanthine Alkaloid Free Isolates Component Amount (+)-catechin 1.6%(−)-epicatechin 38.2% B-2 Dimer 11.0% B-5 Dimer 5.3% C-1 Trimer 9.3%Doubly linked dimers 3.0% Tetramer(s) 4.5% Pentamer-Octamer 24.5%Unknowns and higher oligomers 2.6%

TABLE 5 Relative Concentrations of Procyanidins in Aqueous AcetoneExtracts Component Amount (+)-catechin and 41.9% (−)-epicatechin B-2 andB-5 Dimers 13.9% Trimers 11.3% Tetramers 9.9% Pentamers 7.8% Hexamers5.1% Heptamers 4.2% Octamers 2.8% Nonamers 1.6% Decamers 0.7% Undecamers0.2% Dodecamers <0.1%

FIG. 3 shows several procyanidin structures and FIGS. 4A-4E show therepresentative HPLC chromatograms of the five fractions employed in thefollowing screening for anti-cancer or antineoplastic activity. The HPLCconditions for FIGS. 4A-4E were as follows:

-   -   HPLC Conditions: Hewlett Packard 1090 ternary HPLC System        equipped with HP Model 1046A Programmable Fluorescence Detector.    -   Column: Hewlett Packard 5μ Hypersil ODS (200×2.1 mm) Linear        Gradient of 60% B into A at a flow rate of 0.3 mL/min. B=0.5%        acetic acid in methanol; A=0.5% acetic acid in deionized water.        μ_(ex)=280 nm; μ_(em)=316 nm.

FIG. 15 O shows a representative semi-prep HPLC chromatogram of anadditional 12 fractions employed in the screening for anticancer orantineoplastic activity (HPLC conditions stated above).

Example 6 Anti-Cancer, Anti-Tumor or Antineoplastic

Activity of Cocoa Extracts (Procyanidins)

The MTT (3-[4,5-dimethyl thiazol-2yl]-2,5-diphenyltetrazoliumbromide)—microtiter plate tetrazolium cytotoxicity assay originallydeveloped by Mosmann (1983) was used to screen test samples from Example5. Test samples, standards (cisplatin and chlorambucil) and MTT reagentwere dissolved in 100% DMSO (dimethyl sulfoxide) at a 10 mg/mLconcentration. Serial dilutions were prepared from the stock solutions.In the case of the test samples, dilutions ranging from 0.01 through 100μg/mL were prepared in 0.5% DMSO.

All human tumor cell lines were obtained from the American Type CultureCollection. Cells were grown as mono layers in alpha-MEM containing 10%fetal bovine serum, 100 units/mL penicillin, 100 μg/mL streptomycin and240 units/mL nystatin. The cells were maintained in a humidified, 5% CO₂atmosphere at 37° C.

After trypsinization, the cells are counted and adjusted to aconcentration of 50×10⁵ cells/mL (varied according to cancer cell line).200 μL of the cell suspension was plated into wells of 4 rows of a96-well microtiter plate. After the cells were allowed to attach forfour hours, 2 μL of DMSO containing test sample solutions were added toquadruplicate wells. Initial dose-response finding experiments, usingorder of magnitude test sample dilutions were used to determine therange of doses to be examined. Well absorbencies at 540 nm were thenmeasured on a BIO RAD MP450 plate reader. The mean absorbance ofquadruplicate test sample treated wells was compared to the control, andthe results expressed as the percentage of control absorbance plus/minusthe standard deviation. The reduction of MTT to a purple formazanproduct correlates in a linear manner with the number of living cells inthe well. Thus, by measuring the absorbance of the reduction product, aquantitation of the percent of cell survival at a given dose of testsample can be obtained. Control wells contained a final concentration of1% DMSO.

Two of the samples were first tested by this protocol. Sample MM1represented a very crude isolate of cocoa procyanidins and containedappreciable quantities of caffeine and theobromine. Sample MM2represented a cocoa procyanidin isolate partially purified by gelpermeation chromatography. Caffeine and theobromine were absent in MM2.Both samples were screened for activity against the following cancercell lines using the procedures previously described:

-   -   HCT 116 colon cancer    -   ACHN renal adenocarcinoma    -   SK-5 melanoma    -   A498 renal adenocarcinoma    -   MCF-7 breast cancer    -   PC-3 prostate cancer    -   CAPAN-2 pancreatic cancer

Little or no activity was observed with MM1 on any of the cancer celllines investigated. MM2 was found to have activity against HCT-116, PC-3and ACHN cancer cell lines. However, both MM1 and MM2 were found tointerfere with MTT such that it obscured the decrease in absorbance thatwould have reflected a decrease in viable cell number. This interferencealso contributed to large error bars, because the chemical reactionappeared to go more quickly in the wells along the perimeter of theplate. A typical example of these effects is shown in FIG. 5. At thehigh concentrations of test material, one would have expected to observea large decrease in survivors rather than the high survivor levelsshown. Nevertheless, microscopic examinations revealed that cytotoxiceffects occurred, despite the MTT interference effects. For instance, anIC₅₀ value of 0.5 μg/mL for the effect of MM2 on the ACHN cell line wasobtained in this manner.

These preliminary results, in the inventors' view, required amendment ofthe assay procedures to preclude the interference with MTT. This wasaccomplished as follows. After incubation of the plates at 37° C. in ahumidified, 5% CO₂ atmosphere for 18 hours, the medium was carefullyaspirated and replaced with fresh alpha-MEM media. This media was againaspirated from the wells on the third day of the assay and replaced with100 μL of freshly prepared McCoy's medium. 11 μL of a 5 mg/mL stocksolution of MTT in PBS (Phosphate Buffered Saline) were then added tothe wells of each plate. After incubation for 4 hours in a humidified,5% CO₂ atmosphere at 37° C., 100 μL of 0.04 N HCl in isopropanol wasadded to all wells of the plate, followed by thorough mixing tosolubilize the formazan produced by any viable cells. Additionally, itwas decided to subfractionate the procyanidins to determine the specificcomponents responsible for activity.

The subfractionation procedures previously described were used toprepare samples for further screening. Five fractions representing theareas shown in FIG. 1 and component(s) distribution shown in FIGS. 4A-4Ewere prepared. The samples were coded MM2A through MM2E to reflect theseanalytical characterizations and to designate the absence of caffeineand theobromine.

Each fraction was individually screened against the HCT-116, PC-3 andACHN cancer cell lines. The results indicated that the activity did notconcentrate to any one specific fraction. This type of result was notconsidered unusual, since the components in “active” natural productisolates can behave synergistically. In the case of the cocoaprocyanidin isolate (MM2), over twenty detectable components comprisedthe isolate. It was considered possible that the activity was related toa combination of components present in the different fractions, ratherthan the activity being related to an individual component(s).

On the basis of these results, it was decided to combine the fractionsand repeat the assays against the same cancer cell lines. Severalfraction combinations produced cytotoxic effects against the PC-3 cancercell lines. Specifically, IC₅₀ values of 40 μg/mL each for MM2A and MM2Ecombination, and of 20 μg/mL each for MM2C and MM2E combination, wereobtained. Activity was also reported against the HCT-116 and ACHN celllines, but as before, interference with the MTT indicator precludedprecise observations. Replicate experiments were repeatedly performed onthe HCT-116 and ACHN lines to improve the data. However, these resultswere inconclusive due to bacterial contamination and exhaustion of thetest sample material. FIGS. 6A-6D show the dose-response relationshipbetween combinations of the cocoa extracts and PC-3 cancer cells.

Nonetheless, from this data, it is clear that cocoa extracts, especiallycocoa polyphenols or procyanidins, have significant anti-tumor;anti-cancer or antineoplastic activity, especially with respect to humanPC-3 (prostate), HCT-116 (colon) and ACHN (renal) cancer cell lines. Inaddition, those results suggest that specific procyanidin fractions maybe responsible for the activity against the PC-3 cell line.

Example 7 Anti-Cancer, Anti-Tumor or Antineoplastic

Activity of Cocoa Extracts (Procyanidins)

To confirm the above findings and further study fraction combinations,another comprehensive screening was performed.

All prepared materials and procedures were identical to those reportedabove, except that the standard 4-replicates per test dose was increasedto 8 or 12-replicates per test dose. For this study, individual andcombinations of five cocoa procyanidin fractions were screened againstthe following cancer cell lines.

-   -   PC-3 Prostate    -   KB Nasopharyngeal/HeLa    -   HCT-116 Colon    -   ACHN Renal    -   MCF-7 Breast    -   SK-5 Melanoma    -   A-549 Lung    -   CCRF-CEM T-cell leukemia

Individual screenings consisted of assaying different dose levels(0.01-100 μg/mL) of fractions A, B, C, D, and E (See FIGS. 4A-4E anddiscussion thereof, supra) against each cell line. Combinationscreenings consisted of combining equal dose levels of fractions A+B,A+C, A+D, A+E, B+C, B+D, B+E, C+D, C+E, and D+E against each cell line.The results from these assays are individually discussed, followed by anoverall summary.

A. PC-3 Prostate Cell Line

FIGS. 7A-7H show the typical dose response relationship between cocoaprocyanidin fractions and the PC-3 cell line. FIGS. 7D and 7Edemonstrate that fractions D and E were active at an IC₅₀ value of 75μg/mL. The IC₅₀ values that were obtained from dose-response curves ofthe other procyanidin fraction combinations ranged between 60-80 μg/mLwhen fractions D or E were present. The individual IC₅₀ values arelisted in Table 6.

B. KB Nasopharvngeal/HeLa Cell Line

FIGS. 8A-8H show the typical dose response relationship between cocoaprocyanidin fractions and the KB Nasopharyngeal/HeLa cell line. FIGS. 8Dand 8E demonstrate that fractions D and E were active at an IC₅₀ valueof 75 μg/mL. FIGS. 8F-8H depict representative results obtained from thefraction combination study. In this case, procyanidin fractioncombination A+B had no effect, whereas fraction combinations B+E and D+Ewere active at an IC₅₀ value of 60 μg/mL. The IC₅₀ values that wereobtained from other dose response curves from other fractioncombinations ranged from 60-80 μg/mL when fractions D or E were present.The individual IC₅₀ values are listed in Table 6. These results wereessentially the same as those obtained against the PC-3 cell line.

C. HCT-116 Colon Cell Line

FIG. 9A-9H show the typical dose response relationships between cocoaprocyanidin fractions and the HCT-116 colon cell line. FIGS. 9D and 9Edemonstrate that fraction E was active at an IC₅₀ value of approximately400 μg/mL. This value was obtained by extrapolation of the existingcurve. Note that the slope of the dose response curve for fraction Dalso indicated activity. However, no IC₅₀ value was determined from thisplot, since the slope of the curve was too shallow to obtain a reliablevalue. FIGS. 9F-9H depict representative results obtained from thefraction combination study. In this case, procyanidin fractioncombination B+D did not show appreciable activity, whereas fractioncombinations A+E and D+E were active at IC₅₀ values of 500 μg/mL and 85μg/mL, respectively. The IC₅₀ values that were obtained from doseresponse curves of other fraction combinations averaged about 250 μg/mLwhen fraction E was present. The extrapolated IC₅₀ values are listed inTable 6.

D. ACHN Renal Cell Line

FIG. 10A-10H show the typical dose response relationships between cocoaprocyanidin fractions and the ACHN renal cell line. FIGS. 10A-10Eindicated that no individual fraction was active against this cell line.FIGS. 10F-10H depict representative results obtained from the fractioncombination study. In this case, procyanidin fraction combination B+Cwas inactive, whereas the fraction combination A+E resulted in anextrapolated IC₅₀ value of approximately 500 μg/mL. Dose response curvessimilar to the C+D combination were considered inactive, since theirslopes were too shallow. Extrapolated IC₅₀ values for other fractioncombinations are listed in Table 6.

E. A-549 Lung Cell Line

FIGS. 11A-11H show the typical dose response relationships between cocoaprocyanidin fractions and the A-549 lung cell line. No activity could bedetected from any individual fraction or combination of fractions at thedoses used in the assay. However, procyanidin fractions may nonethelesshave utility with respect to this cell line.

F. SK-5 Melanoma Cell Line

FIG. 12A-12H show the typical dose response relationships between cocoaprocyanidin fractions and the SK-5 melanoma cell line. No activity couldbe detected from any individual fraction or combination of fractions atthe doses used in the assay. However, procyanidin fractions maynonetheless have utility with respect to this cell line.

G. MCF-7 Breast Cell Line

FIGS. 13A-13H show the typical dose response relationships between cocoaprocyanidin fractions and the MCF-7 breast cell line. No activity couldbe detected from any individual fraction or combination of fractions atthe doses used in the assay. However, procyanidin fractions maynonetheless have utility with respect to this cell line.

H. CCRF-CEM T-Cell Leukemia Line

A typical dose response curves were originally obtained against theCCRF-CEM T-cell leukemia line. However, microscopic counts of cellnumber versus time at different fraction concentrations indicated that500 μg of fractions A, B and D effected an 80% growth reduction over afour day period. A representative dose response relationship is shown inFIG. 14.

I. Summary

The IC₅₀ values obtained from these assays are collectively listed inTable 6 for all the cell lines except for CCRF-CEM T-cell leukemia. TheT-cell leukemia data was intentionally omitted from the Table, since adifferent assay procedure was used. A general summary of these resultsindicated that the most activity was associated with fractions D and E.These fractions were most active against the PC-3 (prostate) and KB(nasopharyngeal/HeLa) cell lines. These fractions also evidencedactivity against the HCT-116 (colon) and ACHN (renal) cell lines, albeitbut only at much higher doses. No activity was detected against theMCF-7 (breast), SK-5 (melanoma) and A-549 (lung) cell lines. However,procyanidin fractions may nonetheless have utility with respect to thesecell lines. Activity was also shown against the CCRF-CEM (T-cellleukemia) cell line. It should also be noted that fractions D and E arethe most complex compositionally. Nonetheless, from this data it isclear that cocoa extracts, especially cocoa procyanidins, havesignificant anti-tumor, anti-cancer or antineoplastic activity. TABLE 6IC₅₀ Values for Cocoa Procyanidin Fractions Against Various Cell Lines(IC₅₀ values in μg/mL) A- FRACTION PC-3 KB HCT-116 ACHN MCF-7 SK-5 549 AB C D 90 80 E 75 75 400 A + B A + C 125 100 A + D 75 75 A + E 80 75 500500 B + C B + D 75 80 B + E 60 65 200 C + D 80 75 1000 C + E 80 70 250D + E 80 60 85Values above 100 μg/mL were extrapolated from dose response curves

Example 8 Anti-Cancer, Anti-Tumor or Antineoplastic

Activity of Cocoa Extracts (Procyanidins)

Several additional in vitro assay procedures were used to complement andextend the results presented in Examples 6 and 7.

Method A. Crystal Violet Staining Assay

All human tumor cell lines were obtained from the American Type CultureCollection. Cells were grown as monolayers in IMEM containing 10% fetalbovine serum without antibiotics. The cells were maintained in ahumidified, 5% CO₂ atmosphere at 37° C.

After trypsinization, the cells were counted and adjusted to aconcentration of 1,000-2,000 cells per 100 mL. Cell proliferation wasdetermined by plating the cells (1,000-2,000 cells/well) in a 96 wellmicrotiter plate. After addition of 100 μL cells per well, the cellswere allowed to attach for 24 hours. At the end of the 24 hour period,various cocoa fractions were added at different concentrations to obtaindose response results. The cocoa fractions were dissolved in media at a2 fold concentration and 100 μL of each solution was added in triplicatewells. On consecutive days, the plates were stained with 50 μL crystalviolet (2.5 g crystal violet dissolved in 125 mL methanol, 375 mLwater), for 15 min. The stain was removed and the plate was gentlyimmersed into cold water to remove excess stain. The washings wererepeated two more times, and the plates allowed to dry. The remainingstain was solubilized by adding 100 μL of 0.1 M sodium citrate/50%ethanol to each well. After solubilization, the number of cells werequantitated on an ELISA plate reader at 540 nm (reference filter at 410nm). The results from the ELISA reader were graphed with absorbance onthe y-axis and days growth on the x-axis.

Method B. Soft Agar Cloning Assay

Cells were cloned in soft agar according to the method described byNawata et al. (1981). Single cell suspensions were made in mediacontaining 0.8% agar with various concentrations of cocoa fractions. Thesuspensions were aliquoted into 35 mm dishes coated with mediacontaining 1.0% agar. After 10 days incubation, the number of coloniesgreater than 60 μm in diameter were determined on an Ominicron 3600Image Analysis System. The results were plotted with number of colonieson the y-axis and the concentrations of a cocoa fraction on the x-axis.

Method C. XTT-Microculture Tetrazolium Assay

The XTT assay procedure described by Scudiero et al. (1988) was used toscreen various cocoa fractions. The XTT assay was essentially the sameas that described using the MTT procedure (Example 6) except for thefollowing modifications. XTT((2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-((phenylamino)carbonyl)-2H-tetrazoliumhydroxide) was prepared at 1 mg/mL medium without serum, prewarmed to37° C. PMS was prepared at 5 mM PBS. XTT and PMS were mixed together; 10μL of PMS per mL XTT and 50 μL PMS-XTT were added to each well. After anincubation at 37° C. for 4 hr, the plates were mixed 30 min. on amechanical shaker and the absorbance measured at 450-600 nm. The resultswere plotted with the absorbance on the y-axis and days growth orconcentration on the x-axis.

For methods A and C, the results were also plotted as the percentcontrol as the y-axis and days growth or concentration on the x-axis.

A comparison of the XTT and Crystal Violet Assay procedures was madewith cocoa fraction D & E (Example 3B) against the breast cancer cellline MCF-7 p168 to determine which assay was most sensitive. As shown inFIG. 15A, both assays showed the same dose-response effects forconcentrations >75 μg/mL. At concentrations below this value, thecrystal violet assay showed higher standard deviations than the XTTassay results. However, since the crystal violet assay was easier touse, all subsequent assays, unless otherwise specified, were performedby this procedure.

Crystal violet assay results are presented (FIGS. 15B-15E) todemonstrate the effect of a crude polyphenol extract (Example 2) on thebreast cancer cell line MDA MB231, prostate cancer cell line PC-3,breast cancer cell line MCF-7 p163, and cervical cancer cell line Hela,respectively. In all cases a dose of 250 μg/mL completely inhibited allcancer cell growth over a period of 5-7 days. The Hela cell lineappeared to be more sensitive to the extract, since a 100 μg/mL dosealso inhibited growth. Cocoa fractions from Example 3B were also assayedagainst Hela and another breast cancer cell line SKBR-3. The results(FIGS. 15F and 15G) showed that fraction D & E has the highest activity.As shown in FIGS. 15H and 15I, IC₅₀ values of about 40 μg/mL D & E wereobtained from both cancer cell lines.

The cocoa fraction D & E was also tested in the soft agar cloning assaywhich determines the ability of a test compound(s) to inhibit anchorageindependent growth. As shown in FIG. 15J, a concentration of 100 μg/mLcompletely inhibited colony formation of Hela cells.

Crude polyphenol extracts obtained from eight different cocoa genotypesrepresenting the three horticultural races of cocoa were also assayedagainst the Hela cell line. As shown in FIG. 15K all cocoa varietiesshowed similar dose-response effects. The UIT-1 variety exhibited themost activity against the Hela cell line. These results demonstratedthat all cocoa genotypes possess a polyphenol fraction that elicitsactivity against at least one human cancer cell line that is independentof geographical origin, horticultural race, and genotype.

Another series of assays were performed on crude polyphenol extractsprepared on a daily basis from a one ton scale traditional 5-dayfermentation of Brazilian cocoa beans, followed by a 4-day sun dryingstage. The results shown in FIG. 15L showed no obvious effect of theseearly processing stages, suggesting little change in the composition ofthe polyphenols. However, it is known (Lehrian and Patterson, 1983) thatpolyphenol oxidase (PPO) will oxidize polyphenols during thefermentation stage. To determine what effect enzymatically oxidizedpolyphenols would have on activity, another experiment was performed.Crude PPO was prepared by extracting finely ground, unfermented, freezedried, defatted Brazilian cocoa beans with acetone at a ratio of 1 gmpowder to 10 mL acetone. The slurry was centrifuged at 3,000 rpm for 15min. This was repeated three times, discarding the supernatant each timewith the fourth extraction being poured through a Buchner filteringfunnel. The acetone powder was allowed to air dry, followed by assayaccording to the procedures described by McLord and Kilara, (1983). To asolution of crude polyphenols (100 mg/10 mL Citrate-Phosphate buffer,0.02M, pH 5.5) 100 mg of acetone powder (4,000 units activity/mgprotein) was added and allowed to stir for 30 min. with a stream of airbubbled through the slurry. The sample was centrifuged at 5,000 xg for15 min. and the supernatant extracted 3× with 20 mL ethyl acetate. Theethyl acetate extracts were combined, taken to dryness by distillationunder partial vacuum and 5 mL water added, followed by lyophilization.The material was then assayed against Hela cells and the dose-responsecompared to crude polyphenol extracts that were not enzymaticallytreated. The results (FIG. 15M) showed a significant shift in thedose-response curve for the enzymatically oxidized extract, showing thatthe oxidized products were more inhibitory than their native forms.

Example 9 Antioxidant Activity of Cocoa Extracts

Containing Procyanidins

Evidence in the literature suggests a relationship between theconsumption of naturally occurring antioxidants (Vitamins C, E andBeta-carotene) and a lowered incidence of disease, including cancer(Designing Foods, 1993; Caragay, 1992). It is generally thought thatthese antioxidants affect certain oxidative and free radical processesinvolved with some types of tumor promotion. Additionally, some plantpolyphenolic compounds that have been shown to be anticarcinogenic, alsopossess substantial antioxidant activity (Ho et al., 1992; Huang et al.,1992).

To determine whether cocoa extracts containing procyanidins possessedantioxidant properties, a standard Rancimat method was employed. Theprocedures described in Examples 1, 2 and 3 were used to prepare cocoaextracts which were manipulated further to produce two fractions fromgel permeation chromatography. These two fractions are actually combinedfractions A through C, and D and E (See FIG. 1) whose antioxidantproperties were compared against the synthetic antioxidants BHA and BHT.

Peanut Oil was pressed from unroasted peanuts after the skins wereremoved. Each test compound was spiked into the oil at two levels, ˜100ppm and ˜20 ppm, with the actual levels given in Table 7. 50 μL ofmethanol solubilized antioxidant was added to each sample to aid indispersion of the antioxidant. A control sample was prepared with 50 μLof methanol containing no antioxidant.

The samples were evaluated in duplicate, for oxidative stability usingthe Rancimat stability test at 100° C. and 20 cc/min of air.Experimental parameters were chosen to match those used with the ActiveOxygen Method (AOM) or Swift Stability Test (Van Oosten et al., 1981). Atypical Rancimat trace is shown in FIG. 16. Results are reported inTable 8 as hours required to reach a peroxide level of 100 meq. TABLE 7Concentrations of Antioxidants LEVEL 1 LEVEL 2 SAMPLE ppm ButylatedHydroxytoluene (BHT) 24 120 Butylated Hydroxyanisole (BHA) 24 120 CrudeEthyl Acetate Fraction of Cocoa 22 110 Fraction A-C 20 100 Fraction D-E20 100

TABLE 8 Oxidative Stability of Peanut Oil with Various Antioxidants 20ppm 100 ppm SAMPLE average Control 10.5 ± 0.7 BHT 16.5 ± 2.1 12.5 ± 2.1BHA 13.5 ± 2.1 14.0 ± 1.4 Crude Cocoa Fraction 18.0 ± 0.0 19.0 ± 1.4Fraction A-C 16.0 ± 6.4 17.5 ± 0.0 Fraction D-E 14.0 ± 1.4 12.5 ± 0.7

These results demonstrated increased oxidative stability of peanut oilwith all of the additives tested. The highest increase in oxidativestability was realized by the sample spiked with the crude ethyl acetateextract of cocoa. These results demonstrated that cocoa extractscontaining procyanidins have antioxidant potential equal to or greaterthan equal amounts of synthetic BHA and BHT. Accordingly, the inventionmay be employed in place of BHT or BHA in known utilities of BHA or BHT,for instance as an antioxidant and/or food additive. And, in thisregard, it is noted too that the invention is from an edible source.Given these results, the skilled artisan can also readily determine asuitable amount of the invention to employ in such “BHA or BHT”utilities, e.g., the quantity to add to food, without undueexperimentation.

Example 10 Topoisomerase II Inhibition Study

DNA topoisomerase I and II are enzymes that catalyze the breaking andrejoining of DNA strands, thereby controlling the topological states ofDNA (Wang, 1985). In addition to the study of the intracellular functionof topoisomerase, one of the most significant findings has been theidentification of topoisomerase II as the primary cellular target for anumber of clinically important antitumor compounds (Yamashita et al.,1990) which include intercalating agents (m-AMSA, Adriamycin® andellipticine) as well as nonintercalating epipodophyllotoxins. Severallines of evidence indicate that some antitumor drugs have the commonproperty of stabilizing the DNA—topoisomerase II complex (“cleavablecomplex”) which upon exposure to denaturing agents results in theinduction of DNA cleavage (Muller et al., 1989). It has been suggestedthat the cleavable complex formation by antitumor drugs produces bulkyDNA adducts that can lead to cell death.

According to this attractive model, a specific new inducer of DNAtopoisomerase II cleavable complex is useful as an anti-cancer,anti-tumor or antineoplastic agent. In an attempt to identify cytotoxiccompounds with activities that target DNA, the cocoa procyanidins werescreened for enhanced cytotoxic activity against several DNA—damagesensitive cell lines and enzyme assay with human topoisomerase IIobtained from lymphoma.

A. Decatenation of Kinetoplast DNA by Topoisomerase II

The in vitro inhibition of topoisomerase II decatenation of kinetoplastDNA, as described by Muller et al. (1989), was performed as follows.Nuclear extracts containing topoisomerase II activity were prepared fromhuman lymphoma by modifications of the methods of Miller et al. (1981)and Danks et al. (1988). One unit of purified enzyme was enough todecatenate 0.25 μg of kinetoplast DNA in 30 min. at 34° C. KinetoplastDNA was obtained from the trypanosome Crithidia fasciculata. Eachreaction was carried out in a 0.5 mL microcentrifuge tube containing19.5 μL H₂O, 2.5 μL 10× buffer (1× buffer contains 50 mM tris-HCl, pH8.0, 120 mM KCl, 1 mM MgCl₂, 0.5 mM ATP, 0.5 mM dithiothreitol and 30 μgBSA/mL), 1 μL kinetoplast DNA (0.2 μg), and 1 μL DMSO-containing cocoaprocyanidin test fractions at various concentrations. This combinationwas mixed thoroughly and kept on ice. One unit of topoisomerase wasadded immediately before incubation in a waterbath at 34° C. for 30 min.

Following incubation, the decatenation assay was stopped by the additionof 5 μL stop buffer (5% sarkosyl, 0.0025% bromophenol blue, 25%glycerol) and placed on ice. DNA was electrophoresed on a 1% agarose gelin TAE buffer containing ethidium bromide (0.5 μg/mL). Ultravioletillumination at 310 nm wavelength allowed the visualization of DNA. Thegels were photographed using a Polaroid Land camera.

FIG. 17 shows the results of these experiments. Fully catenatedkinetoplast DNA does not migrate into a 1% agarose gel. Decatenation ofkinetoplast DNA by topoisomerase II generates bands of monomeric DNA(monomer circle, forms I and II) which do migrate into the gel.Inhibition of the enzyme by addition of cocoa procyanidins is apparentby the progressive disappearance of the monomer bands as a function ofincreasing concentration. Based on these results, cocoa procyanidinfractions A, B, D, and E were shown to inhibit topoisomerase II atconcentrations ranging from 0.5 to 5.0 μg/mL. These inhibitorconcentrations were very similar to those obtained for mitoxanthrone andm-AMSA (4′-(9-acridinylamino)methanesulfon-m-anisidide).

B. Drug Sensitive Cell Lines

Cocoa procyanidins were screened for cytotoxicity against severalDNA-damage sensitive cell lines. One of the cell lines was the xrs-6 DNAdouble strand break repair mutant developed by P. Jeggo (Kemp et al.,1984). The DNA repair deficiency of the xrs-6 cell line renders themparticularly sensitive to x-irradiation, to compounds that produce DNAdouble strand breaks directly, such as bleomycin, and to compounds thatinhibit topoisomerase II, and thus may indirectly induce double strandbreaks as suggested by Warters et al. (1991). The cytotoxicity towardthe repair deficient line was compared to the cytotoxicity against a DNArepair proficient CHO line, BR1. Enhanced cytotoxicity towards therepair deficient (xrs-6) line was interpreted as evidence for DNAcleavable double strand break formation.

The DNA repair competent CHO line, BR1, was developed by Barrows et al.(1987) and expresses 0⁶-alkylguanine—DNA—alkyltransferase in addition tonormal CHO DNA repair enzymes. The CHO double strand break repairdeficient line (xrs-6) was a generous gift from Dr. P. Jeggo andco-workers (Jeggo et al., 1989). Both of these lines were grown asmonolayers in alpha-MEM containing serum and antibiotics as described inExample 6. Cells were maintained at 3°7C in a humidified 5% CO₂atmosphere. Before treatment with cocoa procyanidins, cells grown asmonolayers were detached with trypsin treatment. Assays were performedusing the MTT assay procedure described in Example 6.

The results (FIG. 18) indicated no enhanced cytotoxicity towards thexrs-6 cells suggesting that the cocoa procyanidins inhibitedtopoisomerase II in a manner different from cleavable double strandbreak formation. That is, the cocoa procyanidins interact withtopoisomerase II before it has interacted with the DNA to form anoncleavable complex.

Noncleavable complex forming compounds are relatively new discoveries.Members of the anthracyclines, podophyllin alkaloids, anthracenediones,acridines, and ellipticines are all approved for clinical anti-cancer,anti-tumor or antineoplastic use, and they produce cleavable complexes(Liu, 1989). Several new classes of topoisomerase II inhibitors haverecently been identified which do not appear to produce cleavablecomplexes. These include amonafide (Hsiang et al., 1989), distamycin(Fesen et al., 1989), flavanoids (Yamashita et al., 1990), saintopin(Yamashita et al., 1991), membranone (Drake et al., 1989), terpenoids(Kawada et al., 1991), anthrapyrazoles (Fry et al., 1985),dioxopiperazines (Tanabe et al., 1991), and the marine acridine—dercitin(Burres et al., 1989).

Since the cocoa procyanidins inactivate topoisomerase II beforecleavable complexes are formed, they have chemotherapy value eitheralone or in combination with other known and mechanistically definedtopoisomerase II inhibitors. Additionally, cocoa procyanidins alsoappear to be a novel class of topoisomerase II inhibitors, (Kashiwada etal., 1993) and may thus be less toxic to cells than other knowninhibitors, thereby enhancing their utility in chemotherapy.

The human breast cancer cell line MCF-7 (ADR) which expresses a membranebound glycoprotein (gp170) to confer multi-drug resistance (Leonessa etal., 1994) and its parental line MCF-7 p168 were used to assay theeffects of cocoa fraction D & E. As shown in FIG. 19, the parental linewas inhibited at increasing dose levels of fraction D & E, whereas theAdriamycin (ADR) resistant line was less effected at the higher doses.These results show that cocoa fraction D & E has an effect on multi-drugresistant cell lines.

Example 11 Synthesis of Procyanidins

The synthesis of procyanidins was performed according to the proceduresdeveloped by Delcour et al. (1983), with modification. In addition tocondensing (+)-catechin with dihydroquercetin under reducing conditions,(−)-epicatechin was also used to reflect the high concentrations of(−)-epicatechin that naturally occur in unfermented cocoa beans. Thesynthesis products were isolated, purified, analyzed, and identified bythe procedures described in Examples 3, 4 and 5. In this manner, thebiflavanoids, triflavanoids and tetraflavanoids are prepared and used asanalytical standards and, in the manner described above with respect tococoa extracts.

Example 12 Assay of Normal Phase Semi-Preparative Fractions

Since the polyphenol extracts are compositionally complex, it wasnecessary to determine which components were active against cancer celllines for further purification, dose-response assays and comprehensivestructural identification. A normal phase semi preparative HPLCseparation (Example 3B) was used to separate cocoa procyanidins on thebasis of oligomeric size. In addition to the original extract, twelvefractions were prepared (FIGS. 2B and 15 O) and assayed at 100 μg/mL and25 μg/mL doses against Hela and SKBR-3 cancer cell lines to determinewhich oligomer possessed the greatest activity. As shown in FIGS. 20Aand B, fractions 4-11 (pentamer-dodecamer) significantly inhibited HeLaand SKBr-3 cancer cell lines at the 100 μg/mL level. These resultsindicated that these specific oligomers had the greatest activityagainst Hela and SKBR-3 cells. Additionally, normal phase HPLC analysisof cocoa fraction D & E indicated that this fraction, used in previousinvestigations, e.g., Example 7, was enriched with these oligomers.

Example 13 HPLC Purification Methods

Method A. GPC Purification

Procyanidins obtained as in Example 2 were partially purified by liquidchromatography on Sephadex LH 20 (72.5×2.5 cm), using 100% methanol asthe eluting solvent, at a flow rate of 3.5 mL/min. Fractions of theeluent were collected after the first 1.5 hours, and the fractions wereconcentrated by a rotary evaporator, redissolved in water and freezedried. These fractions were referred to as pentamer enriched fractions.Approximately 2.00 g of the extract obtained from Example 2 wassubfractionated in this manner. Results are shown in Table 9. TABLE 9Composition of Fractions Obtained: Tetra- TTrimer mer Others FractionMmonomer DDimer (% (% Pentamer Hexamer Heptamer Octamer Nonamer DecamerUndecamer (% (Time) (% Area) (% Area) Area) Area) (% Area) (% Area) (%Area) (% Area) (% Area) (% Area) (% Area) Area) 1:15 73  8 16  3 ND NDND ND ND ND ND ND 1:44 67 19 10  3  1 tr tr tr tr tr tr tr 2:13 30 29 2411  4  1 tr tr tr tr tr tr 2:42  2 16 31 28 15  6  2 tr tr tr tr tr 3:11 1 12 17 25 22 13  7 2 1 tr tr tr 3:40 tr 18 13 18 20 15 10 5 2 tr tr tr4:09 tr  6  8 17 21 19 14 8 4 2 tr trND = not detectedtr = trace amount

Method B. Normal Phase Separation

Procyanidins obtained as Example 2 were separated purified by normalphase chromatography on Supelcosil LC-Si, 100 Å, 5 μm (250×4.6 mm), at aflow rate of 1.0 mL/min, or, in the alternative, Lichrosphere® Silica100, 100 Å, 5 μm (235×3.2 mm), at a flow rate of 0.5 mL/min. Separationswere aided by a step gradient under the following conditions: (Time, %A, % B); (0, 82, 14), (30, 67.6, 28.4), (60, 46, 50), (65, 10, 86), (70,10, 86). Mobile phase composition was A=dichloromethane; B=methanol; andC=acetic acid:water (1:1). Components were detected by fluorescencewhere λ_(ex)=276 nm and λ_(em)=316 nm, and by UV at 280 nm. Theinjection volume was 5.0 μL (20 mg/mL) of the procyanidins obtained fromExample 2. These results are shown in FIGS. 40A and 40B.

In the alternative, separations were aided by a step gradient under thefollowing conditions: (Time, % A, % B); (0, 76, 20); (25, 46, 50); (30,10, 86). Mobile phase composition was A=dichloromethane; B=methanol; andC=acetic acid: water (1:1). The results are shown in FIGS. 41A and 41B.

Method C. Reverse—Phase Separation

Procyanidins obtained as in Example 2 were separated purified by reversephase chromatography on Hewlett Packard Hypersil ODS 5 μm. (200×2.1 mm),and a Hewlett Packard Hypersil ODS 5 μm guard column (20×2.1 mm). Theprocyanidins were eluted with a linear gradient of 20% B into A in 20minutes, followed by a column wash with 100% B at a flow rate of 0.3mL/min. The mobile phase composition was a degassed mixture of B=1.0%acetic acid in methanol and A=2.0% acetic acid in nanopure water.Components were detected by UV at 280 nm, and fluorescence whereλ_(ex)=276 nm and λ_(em)=316 nm; and the injection volume was 2.0 μL (20mg/mL).

Example 14 HPLC Separation of Pentamer Enriched Fractions

Method A. Semi-Preparative Normal Phase HPLC

The pentamer enriched fractions were further purified bysemi-preparative normal phase HPLC by a Hewlett Packard 1050 HPLC systemequipped with a Millipore—Waters model 480 LC detector set at 254 nm,which was assembled with a Pharmacia Frac-100 Fraction Collector set topeak mode. Separations were effected on a Supelco 5 μm Supelcosel LC-Si,100 Å column (250×10 mm) connected with a Supelco 5μ Supelguard LC-Siguard column (20×4.6 mm). Procyanidins were eluted by a linear gradientunder the following conditions: (Time, % A, % B); (0, 82, 14), (30,67.6, 28.4), (60, 46, 50), (65, 10, 86), (70, 10, 86) followed by a 10minute re-equilibration. Mobile phase composition was A=dichloromethane;B=methanol; and C=acetic acid:water (1:1). A flow rate of 3 mL/min wasused. Components were detected by UV at 254 nm; and recorded on a Kipp &Zonan BD41 recorder. Injection volumes ranged from 100-250 μl of 10 mgof procyanidin extracts dissolved in 0.25 mL 70% aqueous acetone.Individual peaks or select chromatographic regions were collected ontimed intervals or manually by fraction collection for furtherpurification and subsequent evaluation.

-   -   HPLC conditions: 250×100 mm Supelco Supelcosil LC-Si    -   (5 μm) Semipreparative Column    -   20×4.6 mm Supelco Supelcosil LC-Si    -   (5 μm) Guard Column    -   Detector:        -   Waters LC        -   Spectrophotometer Model            -   480@254 nm    -   Flow rate: 3 mL/min.,    -   Column Temperature: ambient,

Injection: 250 μL of pentamer enriched extract acetic acid:waterGradient: CH₂Cl₂ methanol (1:1) 0 82 14 4 30 67.6 28.4 4 60 46 50 4 6510 86 4 70 10 86 4

Method B. Reverse Phase Separation

Procyanidin extracts obtained as in Example 13 were filtered through a0.45μ nylon filter and analyzed by a Hewlett Packard 1090 ternary phaseHPLC system equipped with a Diode Array detector and a HP model 1046AProgrammable Fluorescence Detector. Separations were effected at 45° C.on a Hewlett Packard 5μ Hypersil ODS column (200×2.1 mm). Theprocyanidins were eluted with a linear gradient of 60% B into A followedby a column wash with B at a flow rate of 0.3 mL/min. The mobile phasecomposition was a de-gassed mixture of B=0.5% acetic acid in methanoland A=0.5% acetic acid in nanopure water. Acetic acid levels in A and Bmobile phases can be increased to 2%. Components were detected byfluorescence, where λ_(ex)×276 nm and λ_(em)=316 nm, and by UV at 280nm. Concentrations of (+)-catechin and (−)-epicatechin were determinedrelative to reference standard solutions. Procyanidin levels wereestimated by using the response factor for (−)-epicatechin.

Method C. Normal Phase Separation

Pentamer enriched procyanidin extracts obtained as in Example 13 werefiltered through a 0.45μ nylon filter and analyzed by a Hewlett Packard1090 Series II HPLC system equipped with a HP Model 1046A ProgrammableFluorescence detector and Diode Array detector. Separations wereeffected at 37° C. on a 5μ Phenomenex Lichrosphereo Silica 100 column(250×3.2 mm) connected to a Supelco Supelguard LC-Si 5μ guard column(20×4.6 mm). Procyanidins were eluted by linear gradient under thefollowing conditions: (time, % A, % B); (0, 82, 14), (30, 67.6, 28.4),(60, 46, 50), (65, 10, 86), (70, 10, 86), followed by an 8 minutere-equilibration. Mobile phase composition was A=dichloromethane,B=methanol, and C=acetic acid:water at a volume ratio of 1:1. A flowrate of 0.5 mL/min was used. Components were detected by fluorescence,where λ_(ex)=276 nm and λ_(em)=316 nm or by UV at 280 nm. Arepresentative HPLC chromatogram showing the separation of the variousprocyanidins is shown in FIG. 2 for one genotype. Similar HPLC profileswere obtained from other Theobroma, Herrania and/or their inter or intraspecific crosses.

HPLC conditions:

-   -   250×3.2 mm Phenomenex Lichrosphere® Silica 100    -   column (5μ) 20×4.6 mm Supelco Supelguard LC-Si (5μ) guard column    -   Detectors: Photodiode Array@280 nm    -   Fluorescence λ_(ex)=276 nm; λ_(em)=316 nm    -   Flow rate: 0.5 mL/min.    -   Column temperature: 37° C.

acetic acid: Gradient: CH₂Cl₂ methanol water (1:1) 0 82 14 4 30 67.628.4 4 60 46 50 4 65 10 86 4 70 10 86 4

Method D. Preparative Normal Phase Separation

The pentamer enriched fractions obtained as in Example 13 were furtherpurified by preparative normal phase chromatography by modifying themethod of Rigaud et al., (1993) J. Chrom. 654, 255-260.

Separations were affected at ambient temperature on a 5μ SupelcosilLC-Si 100 Å column (50×2 cm), with an appropriate guard column.Procyanidins were eluted by a linear gradient under the followingconditions: (time, % A, % B, flow rate); (0, 92.5, 7.5, 10); (10, 92.5,7.5, 40); (30, 91.5, 18.5, 40); (145, 88, 22, 40); (150, 24, 86, 40);(155, 24, 86, 50); (180, 0, 100, 50). Prior to use, the mobile phasecomponents were mixed by the following protocol:

Solvent A preparation (82% CH₂Cl₂, 14% methanol, 2% acetic acid, 2%water):

1. Measure 80 mL of water and dispense into a 4 L bottle.

2. Measure 80 mL of acetic acid and dispense into the same 4 L bottle.

3. Measure 560 mL of methanol and dispense into the same 4 L bottle.

4. Measure 3280 mL of methylene chloride and dispense into the 4 Lbottle.

5. Cap the bottle and mix well.

6. Purge the mixture with high purity Helium for 5-10 minutes to degas.

Repeat steps 1-6 two times to yield 8 volumes of solvent A.

Solvent B preparation (96% methanol, 2% acetic acid, 2% water):

1. Measure 80 mL of water and dispense into a 4 L bottle.

2. Measure 80 mL of acetic acid and dispense into the same 4 L bottle.

3. Measure 3840 mL of methanol and dispense 3840 mL of methanol anddispense into the same 4 L bottle.

4. Cap the bottle and mix well.

5. Purge the mixture with high purity Helium for 5-10 minutes to degas.

Repeat steps 1-5 to yield 4 volumes of solvent B. Mobile phasecomposition was A=methylene chloride with 2% acetic acid and 2% water;B=methanol with 2% acetic acid and 2% water. The column load was 0.7 gin 7 mL. components were detected by UV at 254 nm. A typical preparativenormal phase HPLC separation of cocoa procyanidins is shown in FIG. 42.

HPLC Conditions:

Column: 50×2 cm 5μ Supelcosil LC-Si run (ambient temperature.

Mobile Phase:

-   -   A=Methylene Chloride with 2%    -   Acetic Acid and 2% Water.        -   B=Methanol with 2% Acetic Acid and 2% Water.

Gradient/Flow Profile: TIME FLOW RATE (MIN) % A % B (mL/min) 0 92.5 7.510 10 92.5 7.5 40 30 91.5 8.5 40 145 88.0 22.0 40 150 24.0 86.0 40 15524.0 86.0 50 180 0.0 100.0 50

Example 15 Identification of Procyanidins

Procyanidins obtained as in Example 14, method D were analyzed by MatrixAssisted Laser Desorption Ionization-Time of Flight/Mass Spectrometry(MALDI-TOF/MS) using a HP G2025A MALDI-TOF/MS system equipped with aLecroy 9350 500 MHz Oscilloscope. The instrument was calibrated inaccordance with the manufacturer's instructions with a low molecularweight peptide standard (HP Part No. G2051A) or peptide standard (HPPart No. G2052A) with 2,5-dihydroxybenzoic acid (DHB)(HP Part No.G2056A) as the sample matrix. One (1.0) mg of sample was dissolved in500 μL of 70/30 methanol/water, and the sample was then mixed with DHBmatrix, at a ratio of 1:1, 1:10 or 1:50 (sample:matrix) and dried on amesa under vacuum. The samples were analyzed in the positive ion modewith the detector voltage set at 4.75 kV and the laser power set between1.5 and 8 μJ. Data was collected as the sum of a number of single shotsand displayed as units of molecular weight and time of flight. Arepresentative MALDI-TOF/MS is shown in FIG. 22A.

FIGS. 22 and C show MALDI-TOF/MS spectra obtained from partiallypurified procyanidins prepared as described in Example 3, Method A andused for in vitro assessment as described in Examples 6 and 7, and whoseresults are summarized in Table 6. This data illustrates that theinventive compounds described herein were predominantly found infractions D-E, but not A-C.

The spectra were obtained as follows:

The purified D-E fraction was subjected to MALDI-TOF/MS as describedabove, with the exception that the fraction was initially purified bySEP-PACK® C-18 cartridge. Five (5) mg of fraction D-E in 1 mL nanopurewater was loaded onto a pre-equilibrated SEP-PACK® cartridge. The columnwas washed with 5 mL nanopure water to eliminate contaminants, andprocyanidins were eluted with 1 mL 20% methanol. Fractions A-C were useddirectly, as they were isolated in Example 3, Method A, without furtherpurification.

These results confirmed and extended earlier results (see Example 5,Table 3, FIGS. 20A and B) and indicate that the inventive compounds haveutility as sequestrants of cations. In particular, MALDI-TOF/MS resultsconclusively indicated that procyanidin oligomers of n=5 and higher (seeFIGS. 20A and B; and formula under Objects and Summary of the Invention)were strongly associated with anti-cancer activity with the HeLa andSKBR-3 cancer cell line model. Oligomers of n=4 or less were ineffectivewith these models. The pentamer structure apparently has a structuralmotif which is present in it and in higher oligomers which provides theactivity. Additionally, it was observed that the MALDI-TOF/MS datashowed strong M⁺ ions of Na⁺, 2 Na⁺, K⁺, 2 K⁺, Ca⁺⁺, demonstrating theutility as cation sequestrants.

Example 16 Purification of Oligomeric Fractions

Method A. Purification by Semi-Preparative Reverse Phase HPLC

Procyanidins obtained from Example 14, Method A and B and D were furtherseparated to obtain experimental quantities of like oligomers forfurther structural identification and elucidation (e.g., Example 15, 18,19, and 20). A Hewlett Packard 1050 HPLC system equipped with a variablewavelength detector, Rheodyne 7010 injection valve with 1 mL injectionloop was assembled with a Pharmacia FRAC-100 Fraction Collector.Separations were effected on a Phenomenex Ultracarb® 10μ ODS column(250×22.5 mm) connected with a Phenomenex 10μ ODS Ultracarb® (60×10 mm)guard column. The mobile phase composition was A=water; B=methanol usedunder the following linear gradient conditions: (time, % A); (0,85),(60,50), (90,0 and (110,0) at a flow rate of 5 mL/min. Individual peaksor select chromatographic regions were collected on timed intervals ormanually by fraction collection for further evaluation by MALDI-TOF/MSand NMR. Injection loads ranged from 25-100 mg of material. Arepresentative elution profile is shown in FIG. 23 b.

Method B. Modified Semi-Preparative HPLC

Procyanidins obtained from Example 14, Method A and B and D were furtherseparated to obtain experimental quantities of like oligomers forfurther structural identification and elucidation (e.g., Example 15, 18,19, and 20). Supelcosil LC-Si 5μ column (250×10 mm) with a SupelcosilLC-Si 5μ (20×2 mm) guard column. The separations were effected at a flowrate of 3.0 mL/min, at ambient temperature. The mobile phase compositionwas A=dichloromethane; B=methanol; and C=acetic acid:water (1:1); usedunder the following linear gradient conditions: (time, % A, % B); (0,82, 14); (22, 74, 21); (32, 74, 21); (60, 74, 50, 4); (61, 82, 14),followed by column re-equilibration for 7 minutes. Injection volumeswere 60 μL containing 12 mg of enriched pentamer. Components weredetected by UV at 280 nm. A representative elution profile is shown inFIG. 23A.

Example 17 Molecular Modeling of Pentamers

Energy minimized structures were determined by molecular modeling usingDesktop Molecular Modeller, version 3.0, Oxford University Press, 1994.Four representative views of [EC(4→8)]₄-EC (EC=epicatechin) pentamersbased on the structure of epicatechin are shown in FIGS. 24 A-D. Ahelical structure is suggested. In general when epicatechin is the firstmonomer and the bonding is 4→8, a beta configuration results, when thefirst monomer is catechin and the bonding is 4→8, an alpha configurationresults; and, these results are obtained regardless of whether thesecond monomer is epicatechin or catechin (an exception isent-EC(4→8)ent-EC). FIGS. 38A-38P show preferred pentamers, and, FIGS.39A to 39P show a library of stereoisomers up to and including thepentamer, from which other compounds within the scope of the inventioncan be prepared, without undue experimentation.

Example 18 NMR Evaluation of Pyrocyanidins

¹³C NMR spectroscopy was deemed a generally useful technique for thestudy of procyanidins, especially as the phenols usually provide goodquality spectra, whereas proton NMR spectra are considerably broadened.The ¹³C NMR spectra of oligomers yielded useful information for A or Bring substitution patterns, the relative stereochemistry of the C ringand in certain cases, the position of the interflavanoid linkages.Nonetheless, ¹H NMR spectra yielded useful information.

Further, HOHAHA, makes use of the pulse technique to transfermagnetization of a first hydrogen to a second in a sequence to obtaincross peaks corresponding to alpha, beta, gamma or delta protons. COSYis a 2D-Fourier transform NMR technique wherein vertical and horizontalaxes provide ¹H chemical shift and 1D spectra; and a point ofintersection provides a correlation between protons, whereby spin-spincouplings can be determined. HMQC spectra enhances the sensitivity ofNMR spectra of nuclei; other than protons and can reveal cross peaksfrom secondary and tertiary carbons to the respective protons. APT is a¹³C technique used in determining the number of hydrogens present at acarbon. An even number of protons at a carbon will result in a positivesignal, while an odd number of protons at a carbon will result in anegative signal.

Thus ¹³C NMR, ¹H NMR, HOHAHA (homonuclear Hartmann-Hahn), HMQC(heteronuclear multiple quantum coherence), COSY (Homonuclearcorrelation spectroscopy), APT (attached proton test), and XHCORR (avariation on HMQC) spectroscopy were used to elucidate the structures ofthe inventive compounds.

Method A. Monomer

All spectra were taken in deuterated methanol, at room temperature, atan approximate sample concentration of 10 mg/mL. Spectra were taken on aBruker 500 MHZ NMR, using methanol as an internal standard.

FIGS. 44A-E represent the NMR spectra which were used to characterizethe structure of the epicatechin monomer. FIG. 44A shows the ¹H and ¹³Cchemical shifts, in tabular form. FIGS. 44 B-E show ¹H, APT, XHCORR andCOSY spectra for epicatechin.

Similarly, FIGS. 45A-F represent the NMR spectra which were used tocharacterize the structure of the catechin monomer. FIG. 45A shows the¹H and ¹³C chemical shifts, in tabular form. FIGS. 44 B-F show ¹H, ¹³C,APT, XHCORR and COSY spectra for catechin.

Method B. Dimers

All spectra were taken in 75% deuterated acetone in D₂O, using acetoneas an internal standard, and an approximate sample concentration of 10mg/mL.

FIGS. 46A-G represent the spectra which were used to characterize thestructure of the B2 dimer. FIG. 46A shows ¹H and ¹³C chemical shifts, intabular form. The terms T and B indicate the top half of the dimer andthe bottom half of the dimer.

FIGS. 46B and C show the ¹³C and APT spectra, respectively, taken on aBruker 500 MHZ NMR, at room temperature.

FIGS. 46D-G show the ¹H, HMQC, COSY and HOHAHA, respectively, which weretaken on AMZ-360 MHZ NMR at a −7° C. The COSY spectrum was taken using agradient pulse.

FIGS. 47A-G represent the spectra which were used to characterize thestructure of the B5 dimer. FIG. 47A shows the ¹³C and ¹H chemicalshifts, in tabular form.

FIGS. 47B-D show the ¹H, ¹³C and APT, respectively, which were taken ona Bruker 500 MHZ NMR, at room temperature.

FIG. 47E shows the COSY spectrum, taken on an AMX-360, at roomtemperature, using a gradient pulse.

FIGS. 47F and G show the HMQC and HOHAHA, respectively, taken on anAMX-360 MHZ NMR, at room temperature.

Method C. Trimer—Epicatechin/Catechin

All spectra were taken in 75% deuterated acetone in D₂O, at −3° C. usingacetone as an internal standard, on an AMX-360 MHZ NMR, and anappropriate sample concentration of 10 mg/mL.

FIGS. 48A-D represent the spectra which were used to characterize thestructure of the epicatechin/catechin trimer. These figures show ¹H,COSY, HMQC and HOHAHA, respectively. The COSY spectrum was taken using agradient pulse.

Method D. Trimer—All Epicatechin

All spectra were taken in 70% deuterated acetone in D₂O, at −1.8° C.,using acetone as an internal standard, on an AMX-360 MHZ NMR, and anappropriate sample concentration of 10 mg/mL.

FIGS. 49A-D represent the spectra which were used to characterize thestructure of all epicatechin trimer. These figures show ¹H, COSY, HMQCand HOHAHA, respectively. The COSY spectrum was taken using a gradientpulse.

Example 19 Thiolysis of Procyanidins

In an effort to characterize the structure of procyanidins, benzylmercaptan (BM) was reacted with catechin, epicatechin or dimers B2 andB5. Benzyl mercaptan, as well as phloroglucinol and thiophenol, can beutilized in the hydrolysis (thiolysis) of procyanidins in analcohol/acetic acid environment. Catechin, epicatechin or dimer (1:1mixture of B2 and B5 dimers)(2.5 mg) was dissolved in 1.5 mL ethanol,100 μL BM and 50 μL acetic acid, and the vessel (Beckman amino acidanalysis vessel) was evacuated and purged with nitrogen repeatedly untila final purge with nitrogen was followed by sealing the reaction vessel.The reaction vessel was placed in a heat block at 95° C., and aliquotsof the reaction were taken at 30, 60, 120 and 240 minutes. The relativefluorescence of each aliquot is shown in FIGS. 25A-C, representingepicatechin, catechin and dimers, respectively. Higher oligomers aresimilarly thiolyzed.

Example 20 Thiolysis and Desulfurization of Dimers

Dimers B2 and B5 were hydrolyzed with benzylmercaptan by dissolvingdimer (B2 or B5; 1.0 mg) in 600 μl ethanol, 40 μL BM and 20 μL aceticacid. The mixture was heated at 95° C. for 4 hours under nitrogen in aBeckman Amino Acid Analysis vessel. Aliquots were removed for analysisby reverse-phase HPLC, and 75 μL of each of ethanol Raney Nickel andgallic acid (10 mg/mL) were added to the remaining reaction medium in a2 mL hypovial. The vessel was purged under hydrogen, and occasionallyshaken for 1 hour. The product was filtered through a 0.45μ filter andanalyzed by reverse-phase HPLC. Representative elution profiles areshown in FIGS. 26 A and B. Higher oligomers are similarly desulfurized.This data suggests polymerization of epicatechan or catechin andtherefore represents a synthetic route for preparation of inventivecompounds.

Example 21 In Vivo Activity of Pentamer in MDA MB 231 Nude Mouse Model

MDA-MB-231/LCC6 cell line. The cell line was grown in improved minimalessential medium (IMEM) containing 10% fetal bovine serum and maintainedin a humidified, 5% CO₂ atmosphere at 37° C.

Mice. Female six to eight week old NCr nu/nu (athymic) mice werepurchased through NCI and housed in an animal facility and maintainedaccording to the regulations set forth by the United States Departmentof Agriculture, and the American Association for the Accreditation ofLaboratory Animal Care. Mice with tumors were weighed every other day,as well as weekly to determine appropriate drug dosing.

Tumor implantation. MDA-MD-231 prepared by tissue culture was dilutedwith IMEM to 3.3×10⁶ cells/mL and 0.15 mL (i.e. 0.5×10⁶ cells) wereinjected subcutaneously between nipples 2 and 3 on each side of themouse. Tumor volume was calculated by multiplying:length×width×height×0.5. Tumor volumes over a treatment group wereaveraged and Student's t test was used to calculate p values.

Sample preparation. Plasma samples were obtained by cardiac puncture andstored at −70° C. with 15-20 mM EDTA for the purposes of blood chemistrydeterminations. No differences were noted between the control group andexperimental groups.

Fifteen nude mice previously infected with 500,000 cells subcutaneouslywith tumor cell line MDA-MB-231, were randomly separated into threegroups of 5 animals each and treated by intraperitoneal injection withone of: (i) placebo containing vehicle alone (DMSO); (ii) 2 mg/mouse ofpurified pentameric procyanidin extract as isolated in Example 14 methodD in vehicle (DMSO); and (iii) 10 mg/mouse purified pentamericprocyanidin extract as isolated in Example 14, method D in vehicle(DMSO).

The group (iii) mice died within approximately 48 to 72 hours afteradministration of the 10 mg, whereas the group (ii) mice appearednormal. The cause of death of the group (iii) mice was undetermined;and, cannot necessarily be attributed to the administration of inventivecompounds. Nonetheless, 10 mg was considered an upper limit with respectto toxicity.

Treatment of groups (i) and (ii) was repeated once a week, and tumorgrowth was monitored for each experimental and control group. After twoweeks of treatment, no signs of toxicity were observed in the mice ofgroup (ii) and, the dose administered to this group was incrementallyincreased by ½ log scale each subsequent week. The following Tablerepresents the dosages administered during the treatment schedule formice of group (ii): Dose Week (mg/mouse) 1 2 2 2 3 4 4 5 5 5 6 5 7 5

The results of treatment are shown in FIGS. 27A and B and Table 10.TABLE 10 IN VIVO ANTI-CANCER RESULTS % SURVIVAL % SURVIVAL % SURVIVALDAY GROUP (i) GROUP (ii) GROUP (iii) 1 100 100 100 2 100 100 100 3 100100 0 4 100 100 5 100 100 6 100 100 7 100 100 8 100 100 9 100 100 10 100100 11 100 100 12 100 100 13 100 100 14 100 100 15 100 100 16 100 100 17100 100 18 100 100 19 100 100 20 100 100 21 100 100 22 75 100 23 75 10024 75 100 25 75 100 26 75 100 27 75 100 28 75 100 29 50 100 30 50 100 3150 100 32 50 100 33 50 100 34 50 100 35 50 100 36 25 100 37 25 100 38 25100 39 25 100 40 25 100 41 25 100 42 25 100 43 25 80 44 25 80 45 25 8046 25 80 47 25 80 48 25 80 49 25 80 50 25 60 51 25 60 52 25 60 53 25 6054 25 60 55 25 60 56 25 60 57 0 40 58 40 59 40 60 40 61 40 62 40 63 4064 40

These results demonstrate that the inventive fractions and the inventivecompounds indeed have utility in antineoplastic compositions, and arenot toxic in low to medium dosages, with toxicity in higher dosages ableto be determined without undue experimentation.

Example 22 Antimicrobial Activity of Cocoa Extracts

Method A:

A study was conducted to evaluate the antimicrobial activity of crudeprocyanidin extracts from cocoa beans against a variety ofmicroorganisms important in food spoilage or pathogenesis. The cocoaextracts from Example 2, method A were used in the study. An agar mediumappropriate for the growth of each test culture (99 mL) was seeded with1 mL of each cell culture suspension in 0.45% saline (final population10²-10⁴ cfu/mL), and poured into petri dishes. Wells were cut intohardened agar with a #2 cork borer (5 mm diameter). The plates wererefrigerated at 4° C. overnight, to allow for diffusion of the extractinto the agar, and subsequently incubated at an appropriate growthtemperature for the text organism. The results were as follows:

Sample Zone of Inhibition (mm) Extract Concentration B. B. P. B. (mg/mL)sphericus cereus S. aureus aeruginosa subtilis 0 NI NI NI NI NI 25 NI 12NI 11 NI 250 12 20 19 19 11 500 14 21 21 21 13NI = no inhibition

Antimicrobial activity of purified procyanidin extracts from cocoa beanswas demonstrated in another study using the well diffusion assaydescribed above (in Method A) with Staphylococcus aureus as the textculture. The results were as follows:

-   -   cocoa extracts:        -   10 mg/100 μL decaffeinated/detheobrominated        -   acetone extract as in Example 13, method A        -   10 mg/100 μL dimer (99% pure) as in Example 14, method D        -   10 mg/100 μL tetramer (95% pure) as in Example 14, method D        -   10 mg/100 μL hexamer (88% pure) as in Example 14, method D        -   10 mg/100 μL octamer/nonamer (92% pure) as in Example 14,            method D

10 mg/100 μL nonamer & higher (87% pure) as in Example 14, method DSample Zone of Inhibition (mm) 0.45% saline 0 Dimer 33 Tetramer 27Hexamer 24 0.45% saline 0 Octamer 22 Nonamer 20 Decaff./detheo. 26

Method B:

Crude procyanidin extract as in Example 2, method 2 was added in varyingconcentrations to TSB (Trypticase Soy Broth) with phenol red (0.08 g/L),The TSB were inoculated with cultures of Salmonella enteritidis or S.newport (10⁵ cfu/mL), and were incubated for 18 hours at 35° C. Theresults were as follows: S. enteritidis S. Newport 0 mg/mL + + 50 + +100 + + 250 + − 500 − − 750 − −where +=outgrowth, and −=no growth, as evidenced by the change in brothculture from red to yellow with acid production. Confirmation ofinhibition was made by plating from TSB tubes onto XLD plates.

This Example demonstrates that the inventive compounds are useful infood preparation and preservation.

This Example further demonstrates that gram negative and gram positivebacterial growth can be inhibited by the inventive compounds. From this,the inventive compounds can be used to inhibit Helicobacter pylori.Helicobacter pylori has been implicated in causing gastric ulcers andstomach cancer. Accordingly, the inventive compounds can be used totreat or prevent these and other maladies of bacterial origin. Suitableroutes of administration, dosages, and formulations can be determinedwithout undue experimentation considering factors well known in the artsuch as the malady, and the age, weight, sex, general health of thesubject.

Example 23 Halogen-Free Analytical Separation of Extract

Procyanidins obtained from Example 2 were partially purified byAnalytical Separation by Halogen-free Normal Phase Chromatography on 100Å Supelcosil LC-Si 5 μm (250×4.6 mm), at a flow rate of 1.0 mL/min, anda column temperature of 37° C. Separations were aided by a lineargradient under the following conditions: (time, % A, % B); (0, 82, 14);(30, 67.6, 28.4); (60, 46, 50). Mobile phase composition was A=30/70%diethyl ether/Toluene; B=Methanol; and C=acetic acid/water (1:1).Components were detected by UV at 280 nm. A representative elutionprofile is shown in FIG. 28.

Example 24 Effect of Pore Size of Stationary Phase for Normal Phase HPLCSeparation of Procyanidins

To improve the separation of procyanidins, the use of a larger pore sizeof the silica stationary phase was investigated. Separations wereeffected on Silica-300, 5 μm, 300 Å (250×2.0 mm), or, in thealternative, on Silica-1000, 5 μm, 1000 Å (250×2.0 mm). A lineargradient was employed as mobile phase composition was:A=Dichloromethane; B=Methanol; and C=acetic acid/water (1:1). Componentswere detected by fluorescence, wherein λ_(ex)=276 nm and λ_(em)=316 nm,by UV detector at 280 nm. The flow rate was 1.0 mL/min, and the oventemperature was 37° C. A representative chromatogram from threedifferent columns (100 Å pore size, from Example 13, Method D) is shownin FIG. 29. This shows effective pore size for separation ofprocyanidins.

Example 25 Obtaining Desired Procyanidins Via Manipulating Fermentation

Microbial strains representative of the succession associated with cocoafermentation were selected from the M&M/Mars cocoa culture collection.The following isolates were used:

Acetobacter aceti ATCC 15973

Lactobacillus sp. (BH 42)

Candida cruzii (BA 15)

Saccharomyces cerevisiae (BA 13)

Bacillus cereus (BE 35)

Bacillus sphaericus (ME 12)

Each strain was transferred from stock culture to fresh media. Theyeasts and Acetobacter were incubated 72 hours at 26° C. and the bacilliand Lactobacillus were incubated 48 hours at 37° C. The slants wereharvested with 5 mL phosphate buffer prior to use.

Cocoa beans were harvested from fresh pods and the pulp and testaremoved. The beans were sterilized with hydrogen peroxide (35%) for 20seconds, followed by treatment with catalase until cessation ofbubbling. The beans were rinsed twice with sterile water and the processrepeated. The beans were divided into glass jars and processed accordingto the regimens detailed in the following Table: Fermentation WaterEthanol/acid infusate Model Fermentation daily daily transfer tosolutions daily transfer to bench scale model transfer of alcohol andacid fermented pulp fermentation in to fresh corresponding to levelspasteurized on sterile pulp water determined at each stage eachsuccessive coinoculated with of a model pulp day of test strainsfermentation fermentation

The bench scale fermentation was performed in duplicate. All treatmentswere incubated as indicated below:

Day 1: 26° C.

Day 2: 26° C. to 50° C.

Day 3: 50° C.

Day 4: 45° C.

Day 5: 40° C.

The model fermentation was monitored over the duration of the study byplate counts to assess the microbial population and HPLC analysis of thefermentation medium for the production of microbial metabolites. Aftertreatment, the beans were dried under a laminar flow hood to a wateractivity of 0.64 and were roasted at 66° C. for 15 min. Samples wereprepared for procyanidin analysis. Three beans per treatment were groundand defatted with hexane, followed by extraction with anacetone:water:acetic acid (70:29.5:0.5%) solution. The acetone solutionextract was filtered into vials and polyphenol levels were quantified bynormal phase HPLC as in Example 13, method B. The remaining beans wereground and tasted. The cultural and analytical profiles of the modelbench-top fermentation process is shown in FIGS. 30A-C. The procyanidinprofiles of cocoa beans subjected to various fermentation treatments isshown in FIG. 30D.

This Example demonstrates that the invention need not be limited to anyparticular cocoa genotype; and, that by manipulating fermentation, thelevels of procyanidins produced by a particular Theobroma or Herraniaspecies or their inter or intra species specific crosses thereof can bemodulated, e.g., enhanced.

The following Table shows procyanidin levels determined in specimenswhich are representative of the Theobroma genus and their inter andintra species specific crosses. Samples were prepared as in Examples 1and 2 (methods 1 and 2), and analyzed as in Examples 13, method B. Thisdata illustrates that the extracts containing the inventive compoundsare found in Theobroma and Herrania species, and their intra and interspecies specific crosses. Theobroma and Herrania Species ProcyanidinLevels ppm (μg/g) in defatted powder Oligomer Mono- Tetra- Penta- Hexa-SAMPLE mer Dimer Trimer mer mer mer Heptamer Octamer Nonamer DecamerUndecamer Total T. grandiflorum × 3822 3442 5384 4074 3146 2080 850 421348 198 tr⁺ 23,765 T. obovatum 1¹ T. grandiflorum × 3003 4098 5411 39832931 1914 1090 577 356 198 tr 23,561 T. obovatum 2¹ T. grandiflorum ×4990 4980 7556 5341 4008 2576 1075 598 301 144 tr 31,569 T. obovatum 3A¹T. grandiflorum × 3880 4498 6488 4930 3706 2560 1208 593 323 174 tr28,360 T. obovatum 3B¹ T. grandiflorum × 2647 3591 5328 4240 3304 23801506 815 506 249 tr 24,566 T. obovatum 4¹ T. grandiflorum × 2754 38555299 3872 2994 1990 1158 629 359 196 88 23,194 T. obovatum 6¹ T.grandiflorum × 3212 4134 7608 4736 3590 2274 936 446 278 126 ND* 23,750T. obovatum SIN¹ T. obovatum 1¹ 3662 5683 9512 5358 3858 2454 1207 640302 144 ND 32,820 T. grandiflorum 2608 2178 3090 2704 2241 1586 900 484301 148 tr 16,240 TEFFE² T. grandiflorum 4773 4096 5289 4748 3804 2444998 737 335 156 tr 27,380 TEFFE × T. grandiflorum ² T. grandiflorum ×4752 3336 4916 3900 3064 2039 782 435 380 228 ND 23,832 T. subincanum ¹T. obovatum × 3379 3802 5836 3940 2868 1807 814 427 271 136 tr 23,280 T.subincanum ¹ T. speciosum × 902 346 1350 217 152 120 60 tr tr ND ND3,147 T. sylvestris ¹ T. microcarpum ² 5694 3250 2766 1490 822 356 141tr ND ND ND 14,519 T. cacao, SIAL 21,929 10,072 10,106 7788 5311 32421311 626 422 146 tr 60,753 659, t0 T. cacao, SIAL 21,088 9762 9119 70944774 2906 1364 608 361 176 tr 57,252 659, t24 T. cacao, SIAL 20,887 98929474 7337 4906 2929 1334 692 412 302 tr 58,165 659, t48 T. cacao, SIAL9552 5780 5062 3360 2140 1160 464 254 138 tr ND 27,910 659, t96 T.cacao, SIAL 8581 4665 4070 2527 1628 888 326 166 123 tr ND 22,974 659,t120 Pod Rec. 10/96, 869 1295 545 347 175 97 tr *ND ND 3329 Herraniamariae Sample Rec. prior 130 354 151 131 116 51 tr ND ND 933 to 10/96,Herrania mariae*ND = none detected¹sample designated CPATU⁺tr = trace (<50 μg/g)²sample designated ERJON

Example 26 Effect of Procyanidins on NO Method A

The purpose of this study is to establish the relationship betweenprocyanidins (as in Example 14, method D) and NO, which is known toinduce cerebral vascular dilation. The effects of monomers and higheroligomers, in concentrations ranging from 100 μg/mL to 0.1 μg/mL, on theproduction of nitrates (the catabolites of NO), from HUVEC (humanumbilical vein endothelial cells) is evaluated. HUVEC (from Clonetics)is investigated in the presence or absence of each procyanidin for 24 to48 hours. At the end of the experiments, the supernatants are collectedand the nitrate content determined by calorimetric assay. In separateexperiments, HUVEC is incubated with acetylcholine, which is known toinduce NO production, in the presence or absence of procyanidins for 24to 48 hours. At the end of the experiments, the supernatants arecollected and nitrate content is determined by calorimetric assay. Therole of NO is ascertained by the addition of nitroarginine or(1)-N-methyl arginine, which are specific blockers of NO synthase.

Method B. Vasorelaxation of Phenylephrine-Induced Contracted Rat Artery

The effects of each of the procyanidins (100 μg/mL to 0.1 g/mL on therat artery is the target for study of vasorelaxation ofphenylephrine-induced contracted rat artery. Isolated rat artery isincubated in the presence or absence of procyanidins (as in Example 14,method D) and alteration of the muscular tone is assessed by visualinspection. Both contraction or relaxation of the ray artery isdetermined. Then, using other organs, precontraction of the isolated ratartery is induced upon addition of epinephrine. Once the contraction isstabilized, procyanidins are added and contraction or relaxation of therat artery is determined. The role of NO is ascertained by the additionof nitroarginine or (1)-N-methyl arginine. The acetylcholine-inducedrelaxation of NO, as it is effected by phenylephrine-precontracted rataorta is shown in FIG. 31.

Method C. Induction of Hypotension in the Rat

This method is directed to the effect of each procyanidin (as in Example14, method D) on blood pressure. Rats are instrumented in order tomonitor systolic and diastolic blood pressure. Each of the procyanidinsare injected intravenously (dosage range=100-0.1 μg/kg), and alterationof blood pressure is assessed. In addition, the effect of eachprocyanidin on the alteration of blood pressure evoked by epinephrine isdetermined. The role of NO is ascertained by the addition ofnitroarginine or (1)-N-methyl arginine.

These studies, together with next Example, illustrate that the inventivecompounds are useful in modulating vasodilation, and are further usefulwith respect to modulating blood pressure or addressing coronaryconditions, and migraine headache conditions.

Example 27 Effects of Cocoa Polyphenols on Satiety

Using blood glucose levels as an indicator for the signal events whichoccur in vivo for the regulation of appetite and satiety, a series ofsimple experiments were conducted using a healthy male adult volunteerage 48 to determine whether cocoa polyphenols would modulate glucoselevels. Cocoa polyphenols were partially purified from Brazilian cocoabeans according to the methods described by Clapperton et al. (1992).This material contained no caffeine or theobromine. Fasting bloodglucose levels were analyzed on a timed basis after ingestion of 10 fl.oz of Dexicola 75 (caffeine free) Glucose tolerance test beverage(Curtin Matheson 091-421) with and without 75 mg cocoa polyphenols. Thislevel of polyphenols represented 0.1% of the total glucose of the testbeverage and reflected the approximate amount that would be present in astandard 100 g chocolate bar. Blood glucose levels were determined byusing the Accu-Chek III blood glucose monitoring system (BoehringerMannheim Corporation). Blood glucose levels were measured beforeingestion of test beverage, and after ingestion of the test beverage atthe following timed intervals: 15, 30, 45, 60, 75, 90, 120 and 180minutes. Before the start of each glucose tolerance test, high and lowglucose level controls were determined. Each glucose tolerance test wasperformed in duplicate. A control test solution containing 75 mg cocoapolyphenols dissolved in 10 fl. oz. distilled water (no glucose) wasalso performed.

Table 11 below lists the dates and control values obtained for eachglucose tolerance experiment performed in this study. FIG. 32 representsplots of the average values with standard deviations of blood glucoselevels obtained throughout a three hour time course. It is readilyapparent that there is a substantial increase in blood sugar levels wasobtained after ingestion of a test mixture containing cocoa polyphenols.The difference between the two principal glucose tolerance profilescould not be resolved by the profile obtained after ingestion of asolution of cocoa polyphenols alone. The addition of cocoa polyphenolsto the glucose test beverage raised the glucose tolerance profilesignificantly. This elevation in blood glucose levels is within therange considered to be mildly diabetic, even though the typical glucosetolerance profile was considered to be normal (Davidson, I. et al., Eds.Todd—Sandford Clinical Diagnosis by Laboratory Methods 14^(th) edition;W.B. Saunders Co.; Philadelphia, Pa. 1969 Ch. 10, pp. 550-9). Thissuggests that the difference in additional glucose was released to thebloodstream, from the glycogen stores, as a result of the inventivecompounds. Thus, the inventive compounds can be used to modulate bloodglucose levels when in the presence of sugars. TABLE 11 GlucoseTolerance Test Dates and Control Results HIGH LOW WEEK DESCRIPTIONCONTROL^(a) CONTROL^(b) 0 Glucose Tolerance 265 mg/dL 53 mg/dL 1 GlucoseTolerance with 310 68 0.1% polyphenols 2 Glucose Tolerance 315 66 4Glucose Tolerance with 325 65 0.1% polyphenols 5 0.1% polyphenols 321 66^(a)= Expected range: 253-373 mg/dL^(b)= Expected range: 50-80 mg/dL

The subject also experienced a facial flush (erythema) andlightheadedness following ingestion of the inventive compounds,indicating modulation of vasodilation.

The data presented in Tables 12 and 13 illustrates the fact thatextracts of the invention pertaining to cocoa raw materials andcommercial chocolates, and inventive compounds contained therein can beused as a vehicle for pharmaceutical, veterinary and food sciencepreparations and applications. TABLE 12 Procyanidin Levels in CommercialChocolates μg/g Heptamers and Sample Monomers Dimers Trimers TetramersPentamers Hexamers Higher Total Brand 1 366 166 113 59 56 23 18 801Brand 2 344 163 111 45 48 ND* ND 711 Brand 3 316 181 100 41 40 7 ND 685Brand 4 310 122 71 27 28 5 ND 563 Brand 5 259 135 90 46 29 ND ND 559Brand 6 308 139 91 57 47 14 ND 656 Brand 7 196 98 81 58 54 19 ND 506Brand 8 716 472 302 170 117 18 ND 1,795 Brand 9 1,185 951 633 298 173 2521 3,286 Brand 10 1,798 1,081 590 342 307 93 ND 4,211 Brand 11 1,101 746646 372 347 130 75 3,417 Brand 12 787 335 160 20 10 8 ND 1,320ND* = None detected.

TABLE 13 Procyanidin Levels in Cocoa Raw Materials μg/g Heptamers SampleMonomers Dimers Trimers Tetramers Pentamers Hexamers and Higher TotalUnfermented 13,440 6,425 6,401 5,292 4,236 3,203 5,913 44,910 Fermented2,695 1,538 1,362 740 470 301 277 7,383 Roasted 2,656 1,597 921 337 164ND* ND 5,675 Choc. Liquor 2,805 1,446 881 442 184 108 ND 5,866 CocoaHulls 114 53 14 ND ND ND ND 181 Cocoa Powder 1% Fat 506 287 112 ND ND NDND 915 Cocoa Powder 11% Fat 1,523 1,224 680 46 ND ND ND 3,473 Red DutchCocoa Powder, 1,222 483 103 ND ND ND ND 1,808 pH 7.4, 11% fat Red DutchCocoa Powder, 168 144 60 ND ND ND ND 372 pH 8.2, 23% fatND* = None detected.

Example 28 The Effect of Procyanidins on Cyclooxygenase 1 & 2

The effect of procyanidins on cyclooxygenase 1 & 2 (COX1/COX2)activities was assessed by incubating the enzymes, derived from ramseminal vesicle and sheep placenta, respectively, with arachidonic acid(5 μM) for 10 minutes at room temperature, in the presence of varyingconcentrations of procyanidin solutions containing monomer to decamerand procyanidin mixture. Turnover was assessed by using PGE2 EIA kitsfrom Interchim (France). Indomethacin was used as a reference compound.The results are presented in the following Table, wherein the IC₅₀values are expressed in units of μM (except for S11, which represents aprocyanidin mixture prepared from Example 13, Method A and where thesamples S1 to S10 represent sequentially procyanidin oligomers (monomerthrough decamer) as in Example 14, Method D, and IC₅₀ is expressed inunits of mg/mL). IC₅₀ IC₅₀ RATIO IC₅₀ SAMPLE # COX-1 (*) COX-2 (*)COX2/COX1 1 0.074 0.197 2.66 2 0.115 0.444 3.86 3 0.258 0.763 2.96 40.154 3.73 24.22 5 0.787 3.16 4.02 6 1.14 1.99 1.75 7 1.89 4.06 2.15 82.25 7.2 3.20 9 2.58 2.08 0.81 10  3.65 3.16 0.87 11  0.0487 0.0741 1.52Indomethacin 0.599 13.5 22.54(*) expressed as uM with the exception of sample 11, which is mg/mL.

The results of the inhibition studies are presented in FIGS. 33 A and B,which shows the effects of Indomethacin on COX1 and COX2 activities.FIGS. 34 A and B shows the correlation between the degree ofpolymerization of the procyanidin and IC₅₀ with COX1 and COX2; FIG. 35shows the correlation between IC₅₀ values on COX1 and COX2. And, FIGS.36 A through Y show the IC₅₀ values of each sample (S1-S11) with COX1and COX2.

These results indicate that the inventive compounds have analgesic,anti-coagulant, and anti-inflammatory utilities. Further, COX2 has beenlinked to colon cancer. Inhibition of COX2 activity by the inventivecompounds illustrates a plausible mechanism by which the inventivecompounds have antineoplastic activity against colon cancer.

COX1 and COX2 are also implicated in the synthesis of prostaglandins.Thus, the results in this Example also indicate that the inventivecompounds can modulate renal functions, immune responses, fever, pain,mitogenesis, apoptosis, prostaglandin synthesis, ulceration (e.g.,gastric), and reproduction. Note that modulation of renal function canaffect blood pressure; again implicating the inventive compounds inmodulating blood pressure, vasodilation, and coronary conditions (e.g.,modulation of angiotensin, bradykinin).

Reference is made to Seibert et al., PNAS USA 91:12013-12017 (December,1994), Mitchell et al., PNAS USA 90:11693-11697 (December 1994), Dewittet al., Cell 83:345-348 (Nov. 3, 1995), Langenbach et al., Cell83:483-92 (Nov. 3, 1995) and Sujii et al., Cell 83:493-501 (Nov. 3,1995), Morham et al., Cell 83:473-82 (Nov. 3, 1995).

Reference is further made to Examples 9, 26, and 27. In Example 9, theanti-oxidant activity of inventive compounds is shown. In Example 26,the effect on NO is demonstrated. And, Example 27 provides evidence of afacial vasodilation. From the results in this Example, in combinationwith Examples 9, 26 and 27, the inventive compounds can modulate freeradical mechanisms driving physiological effects. Similarly,lipoxygenase mediated free radical type reactions biochemically directedtoward leukotriene synthesis can be modulated by the inventivecompounds, thus affecting subsequent physiological effects (e.g.,inflammation, immune response, coronary conditions, carcinogenicmechanisms, fever, pain, ulceration).

Thus, in addition to having analgesic properties, there may also be asynergistic effect by the inventive compounds when administered withother analgesics. Likewise, in addition to having antineoplasticproperties, there may also be a synergistic effect by the inventivecompounds when administered with other antineoplastic agents.

Example 29 Circular Dichroism/Study of Procyanidins

CD studies were undertaken in an effort to elucidate the structure ofpurified procyanidins as in Example 14, Method D. The spectra werecollected at 25° C. using CD spectrum software AVIV 60DS V4.1f.

Samples were scanned from 300 nm to 185 nm, every 1.00 nm, at 1.50 nmbandwidth. Representative CD spectra are shown in FIGS. 43A through G,which show the CD spectra of dimer through octamer.

These results are indicative of the helical nature of the inventivecompounds.

Example 30 Inhibitory Effects of Cocoa Procyanidins on HelicobacterPylori and Staphylococcus aureus

A study was conducted to evaluate the antimicrobial activity ofprocyanidin oligomers against Helicobacter pylori and Staphylococcusaureus. Pentamer enriched material was prepared as described in Example13, Method A and analyzed as described in Example 14, Method C, where89% was pentamer, and 11% was higher oligomers (n is 6 to 12). Purifiedpentamer (96.3%) was prepared as described in Example 14, Method D.

Helicobacter pylori and Staphylococcus aureus were obtained from theAmerican Type Culture Collection (ATCC). For H. pylori, the vial wasrehydrated with 0.5 mL Trypticase Soy broth and the suspensiontransferred to a slant of fresh TSA containing 5% defibrinated sheepblood. The slant was incubated at 37° C. for 3 to 5 days undermicroaerophilic conditions in anaerobic jars (5 to 10% carbon dioxide;CampyPakPlus, BBL). When good growth was established in the pool ofbroth at the bottom of the slant, the broth was used to inoculateadditional slants of TSA with sheep blood. Because viability decreasedwith continued subculturing, the broth harvested from the slants waspooled and stored at −80° C. Cultures for assay were used directly fromthe frozen vials. The S. aureus culture was maintained on TSA slants andtransferred to fresh slants 24 h prior to use. A cell suspension of eachculture was prepared (H. pylori, 10⁸ to 10⁹ cfu/mL; S. aureus 10⁶ to 10⁷cfu/mL) and 0.5 mL spread onto TSA plates with 5% sheep blood. Standardassay disks (Difco) were dipped into filter sterilized, serial dilutionsof pentamer (23 mg/mL into sterile water). The test disks and the blankcontrol disks (sterile water) were placed on the inoculated plates.Control disks containing 80 ug metronidazole (inhibitory to H. pylori)or 30 ug vancomycin (inhibitory to S. aureus) (BBL Sensidiscs) were alsoplaced on the appropriate set of plates. The H. pylori inoculated plateswere incubated under microaerophilic conditions. The S. aureus set wasincubated aerobically. Zones of inhibition were measured followingoutgrowth. TABLE 14 Bioassays with pentamer against Helicobacter pyloriand Staphylocuccus aureus Pentamer Enriched Fraction S. aureus H. pylori(mg/ml) Inhibition (mm) Inhibition (mm)  0 NI NI 15  0 10 31 10 10 62 1111 125  13 13 250  15 13 Vancomycin standard 15 — Metronidazole standard— 11 96% pure pentamer 15 11NI = no inhibition

Example 31 NO Dependent Hypotension in the Guinea Pig

The effect of five cocoa procyanidin fractions on guinea pig bloodpressure were investigated. Briefly, guinea pigs (approximately 400 gbody weight; male and female) were anesthetized upon injection of 40mg/kg sodium pentobarbital. The carotid artery was cannulated formonitoring of the arterial blood pressure. Each of the five cocoaprocyanidin fractions was injected intravenously (dose range 0.1mg/kg-100 mg/kg) through the jugular vein. Alterations of blood pressurewere recorded on a polygraph. In these experiments, the role of NO wasascertained by the administration of L-N-methylarginine (1 mg/kg) tenminutes prior to the administration of cocoa procyanidin fractions.

Cocoa procyanidin fractions were prepared and analyzed according to theprocedures described in U.S. Pat. No. 5,554,645, hereby incorporatedherein by reference.

Fraction A: Represents a preparative HPLC fraction comprised ofmonomers-tetramers. HPLC analysis revealed the following composition:Monomers 47.2% Dimers 23.7 Trimers 18.7 Tetramers 10.3

Fraction B: Represents a preparative HPLC fraction comprised ofpentamers-decamers. HPLC analysis revealed the following composition:Pentamers 64.3% Hexamers 21.4 Heptamers 7.4 Octamers 1.9 Nonamers 0.9Decamers 0.2

Fraction C: Represents an enriched cocoa procyanidin fraction used inthe preparation of Fractions A and B (above). HPLC analysis revealed thefollowing composition: Monomers 34.3% Dimers 17.6 Trimers 16.2 Tetramers12.6 Pentamers 8.5 Hexamers 5.2 Heptamers 3.1 Octamers 1.4 Nonamers 0.7Decamers 0.3Fraction D: Represents a procyanidin extract prepared from a milkchocolate. HPLC analysis revealed a composition similar to that listedin the Table 12 for Brand 8. Additionally, caffeine 10% and theobromine6.3% were present.Fraction E: Represents a procyanidin extract prepared from a darkchocolate prepared with alkalized liquor. HPLC analysis revealed acomposition similar to that listed in the Table 12 for Brand 12.Additionally, caffeine 16.0% and theobromine 5.8% were present.

In three separate experiments, the effects of administering 10 mg/kgcocoa procyanidin fractions on arterial blood pressure of anesthetizedguinea pigs was investigated. Upon intravenous injection, procyanidinfractions A and E evoked a decrease in blood pressure of about 20%. Thisdecrease was only marginally different from that obtained from a solvent(DMSO) control (15±5%, n=5). In contrast, procyanidin fractions B, C andD (10 mg/kg) induced marked decreases in blood pressure, up to 50-60%for C. In these experiments the order of hypotensive effect was asfollows: C>B>D>>A=E.

Typical recordings of blood pressure elicited after injection ofprocyanidin fractions appear in FIG. 50A for fraction A and FIG. 50B forfraction C. FIG. 51 illustrates the comparative effects on bloodpressure by these fractions.

The possible contribution of NO in the hypotension in the guinea piginduced by administration of fraction C was analyzed using L-N-methylarginine (LNMMA). This pharmacological agent inhibits the formation ofNO by inhibiting NO synthase. L-NMMA was administered at the dose of 1mg/kg, ten minutes prior to injection of the cocoa procyanidinfractions. Treatment of the animals with L-NMMA completely blocked thehypotension evoked by the procyanidin fraction C. Indeed, followingtreatment with this inhibitor, the alterations of blood pressureproduced by fraction C were similar to those noted with solvent alone.

Example 32 Effect of Cocoa Procyanidin Fractions on NO Production inHuman Umbilical Vein Endothelial Cells

Human umbilical vein endothelial cells (HUVEC) were obtained fromClonetics and cultures were carried out according to the manufacturer'sspecifications. HUVEC cells were seeded at 5,000 cells/cm² in 12-wellplates (Falcon). After the third passage under the same conditions, theywere allowed to reach confluence. The supernatant was renewed with freshmedium containing defined concentrations of bradykinin (25, 50 and 100nM) or cocoa procyanidin fractions A-E (100 μg/mL) as described inexample 31. The culture was continued for 24 hr. and the cell freesupernatants were collected and stored frozen prior to assessment of NOcontent as described below. In selected experiments, the NO synthase(NOS) antagonist, Nω-nitro-L-arginine methyl ester (L-NAME, 10 μM) wasadded to assess the involvement of NOS in the observed NO production.

HUVEC NO production was estimated by measuring nitrite concentration inthe culture supernatant by the Griess reaction. Griess reagent was 1%sulfanilamide, 0.1% N-(1-naphthyl)-ethylenediamine dihydrochloride.Briefly, 50 μL aliquots were removed from the various supernatants inquadruplicate and incubated with 150 μL of the Griess reagent. Theabsorbency at 540 nm was determined in a multiscan (LabsystemsMultiskans MCC/340) apparatus. Sodium nitrite was used at definedconcentrations to establish standard curves. The absorbency of themedium without cells (blank) was subtracted from the value obtained withthe cell containing supernatants.

FIG. 53 illustrates the effect of bradykinin on NO production by HUVECwhere a dose dependent release of NO was observed. The inhibitor L-NAMEcompletely inhibited the bradykinin induced NO release.

FIG. 54 illustrates the effect of the cocoa procyanidin fractions on NOproduction by HUVEC cells. Fractions B, C and D induced a moderate butsignificant amount of NO production by HUVEC. By far, Fraction C was themost efficient fraction to induce NO formation as assessed by theproduction of nitrites, while Fraction E was nearly ineffective. Theeffect of Fraction C on NO production was dramatically reduced in thepresence of L-NAME. Interestingly, Fractions B, C and D contained higheramounts of procyanidin oligomers than Fractions A and E. Adistinguishing difference between Fractions D and E was that E wasprepared from a dark chocolate which used alkalized cocoa liquor as partof the chocolate recipe. Alkalization leads to a base catalyzedpolymerization of procyanidins which rapidly depletes the levels ofthese compounds. An analytical comparison of procyanidin levels found inthese types of chocolate appear in the Table 12, where Brand 12 is adark chocolate prepared with alkalized cocoa liquor and Brand 11 is atypical milk chocolate. Thus, extracts obtained from milk chocolatescontain high proportions of procyanidin oligomers which are capable ofinducing NO. The addition of the NO inhibitor L-NMMA to the Fraction Csample clearly led to the inhibition of NO. The results obtained fromthe procyanidin fractions were consistent to those observed with thebradykinin induced NO experiment (see FIG. 53).

As in the case of the HUVEC results, cocoa procyanidin fraction Celicited a major hypotensive effect in guinea pigs, whereas fractions Aand E were the least effective. Again, the presence of high molecularweight procyanidin oligomers were implicated in the modulation of NOproduction.

Example 33 Effect of Cocoa Procyanidin Fractions on Macrophage NOProduction

Fresh, human heparinized blood (70 mL) was added with an equal volume ofphosphate buffer saline (PBS) at room temperature. A Ficoll-Hypaquesolution was layered underneath the blood-PBS mixture using a 3 mLFicoll-Hypaque to 10 mL blood-PBS dilution ratio. The tubes werecentrifuged for 30 minutes at 2,000 rpm at 18-20° C. The upper layercontaining plasma and platelets was discarded. The mononuclear celllayer was transferred to another centrifuge tube and the cells werewashed 2× in Hanks balanced saline solution. The mononuclear cells wereresuspended in complete RPMI 1640 supplemented with 10% fetal calfserum, counted and the viability determined by the trypan blue exclusionmethod. The cell pellet was resuspended in complete RPMI 1640supplemented with 20% fetal calf serum to a final concentration of 1×10⁶cells/mL. Aliquots of the cell suspension were plated into a 96 wellculture plate and rinsed 3× with RPMI 1640 supplemented with 10% fetalcalf serum and the nonadherent cells (lymphocytes) were discarded.

These cells were incubated for 48 hours in the presence or absence offive procyanidin fractions described in Example 31. At the end of theincubation period, the culture media were collected, centrifuged andcell free supernatants were stored frozen for nitrate assaydeterminations.

Macrophage NO production was determined by measuring nitriteconcentrations by the Greiss reaction. Greiss reagent was 1%sulfanilamide, 0.1% N-(1-naphthyl)-ethylenediamine dihydrochloride.Briefly, 50 μL aliquots were removed from the supernatants inquadruplicate and incubated with 150 μL of the Greiss reagent. Theabsorbency at 540 nm was determined in a multiscan (LabsystemsMultiskans MCC/340) apparatus. Sodium nitrite was used at definedconcentrations to establish standard curves. The absorbency of themedium without cells (blank) was subtracted from the value obtained withthe cell containing supernatants.

In a separate experiment, macrophages were primed for 12 hours in thepresence of 5 U/mL gamma-interferon and then stimulated with 10 μg/mLLPS for the next 36 hours in the presence or absence of 100 μg/mL of thefive procyanidin fractions.

FIG. 55 indicates that only procyanidin fraction C, at 100 μg/mL, couldinduce NO production by monocytes/macrophages. Basal NO production bythese cells was undetectable and no nitrite could be detected in any ofthe cocoa procyanidin fractions used at 100 μg/mL. FIG. 56 indicatesthat procyanidin fractions A and D enhanced LPS-induced NO production byγ-interferon primed monocytes/macrophages. Procyanidin fraction C wasmarginally effective, since LPS-stimulated monocytes/macrophagescultured in the absence of procyanidin fractions produced only 4μmole/10⁵ cells/48 hours. γ-Interferon alone was ineffective in inducingNO.

Collectively, these results demonstrate that mixtures of the inventivecompounds used at specific concentrations are capable of inducingmonocyte/macrophage NO production both independent and dependent ofstimulation by LPS or cytokines.

From the foregoing, it is clear that the extract and cocoa polyphenols,particularly the inventive compounds, as well as the compositions,methods, and kits, of the invention have significant and numerousutilities.

The antineoplastic utility is clearly demonstrated by the in vivo and invitro data herein and shows that inventive compounds can be used insteadof or in conjunction with conventional antineoplastic agents.

The inventive compounds have antioxidant activity like that of BHT andBHA, as well as oxidative stability. Thus, the invention can be employedin place of or in conjunction with BHT or BHA in known utilities of BHAand BHT, such as an antioxidant, for instance, an antioxidant; foodadditive.

The invention can also be employed in place of or in conjunction withtopoisomerase II-inhibitors in the presently known utilities therefor.

The inventive compounds can be used in food preservation or preparation,as well as in preventing or treating maladies of bacterial origin.Simply the inventive compounds can be used as an antimicrobial.

The inventive compounds can also be used as a cyclo-oxygenase and/orlipoxygenase, NO or NO-synthase, or blood or in vivo glucose modulator,and are thus useful for treatment or prevention or modulation of pain,fever, inflammation coronary conditions, ulceration, carcinogenicmechanisms, vasodilation, as well as an analgesic, anti-coagulantanti-inflammatory and an immune response modulator.

Further, the invention comprehends the use of the compounds or extractsas a vehicle for pharmaceutical preparations. Accordingly, there aremany compositions and methods envisioned by the invention. For instance,antioxidant or preservative compositions, topoisomerase II-inhibitingcompositions, methods for preserving food or any desired item such asfrom oxidation, and methods for inhibiting topoisomerase II. Thecompositions can comprise the inventive compounds. The methods cancomprise contacting the food, item or topoisomerase II with therespective composition or with the inventive compounds. Othercompositions, methods and embodiments of the invention are apparent fromthe foregoing.

In this regard, it is mentioned that the invention is from an ediblesource and, that the activity in vitro can demonstrate at least someactivity in vivo; and from the in vitro and in vivo data herein, doses,routes of administration, and formulations can be obtained without undueexperimentation.

Example 34 Micellar Electrokinetic Capillary Chromatography of CocoaProcyanidins

A rapid method was developed using micellar electrokinetic capillarychromatography (MECC) to separate procyanidin oligomers. The method is amodification of that reported by Delgado et al., 1994. The MECC methodrequires only 12 minutes to achieve the same separation as that obtainedby a 70 minute normal phase HPLC analysis. FIG. 57 represents a MECCseparation of cocoa procyanidins obtained by Example 2.

MECC Conditions:

The cocoa procyanidin extract was prepared by the method described inExample 2 and dissolved at a concentration of 1 mg/mL in MECC bufferconsisting of 200 mM boric acid, 50 mM sodium dodecyl sulfate(electrophoresis pure) and NaOH to adjust to pH=8.5.

The sample was passed through a 0.45 um filter and electrophoresed usinga Hewlett Packard HP-3D CZE System operated at the following conditions:

Inlet buffer: Run buffer as described above

Outlet buffer: Run buffer as described above

Capillary: 50 cm×75 um i.d. uncoated fused silica

Detection: 200 nm, with Diode Array Detector

Injection: 50 mBar for 3 seconds (150 mBar sec)

Voltage: 6 watts

Amperage: System limit (<300 uA)

Temperature: 25° C.

Capillary Condition: 5 min flush with run buffer before and after eachrun.

This method can be modified by profiling temperature, pressure, andvoltage parameters, as well as including organic modifiers and chiralselective agents in the run buffer.

Example 35 MALDI—TOF/MS Analysis of Procyanidin Oligomers with MetalSalt Solutions

A series of MALDI-TOF/MS analyses were performed on trimers combinedwith various metal salt solutions to determine whether cation adducts ofthe oligomer could be detected. The significance of the experiment wasto provide evidence that the procyanidin oligomers play a physiologicalrole in vitro and in vivo by sesquestering or delivering metal cationsimportant to physiological processes and disease.

The method used was as described in Example 15. Briefly, 2 uL of 10 mMsolutions of zinc sulfate dihydrate, calcium chloride, magnesiumsulfate, ferric chloride hexahydrate, ferrous sulfate heptahydrate, andcupric sulfate were individually combined with 4 uL of a trimer (10mg/mL) purified to apparent homogeneity as described in Example 14, and44 uL of DHB added.

The results (FIGS. 58A-F) showed [Metal-Trimer+H]⁺ ions for copper andiron (ferrous and ferric) whose m/z values matched ±1 amu standarddeviation value for the theoretical calculated masses. The[Metal-Trimer+H]⁺ masses for calcium and magnesium could not beunequivocally resolved from the [Metal-Trimer+H]⁺ masses for sodium andpotassium, whose m/z values were within the ±1 amu standard deviationvalues. No [Zn⁺²—Trimer+H]⁺ ion could be detected. Since some of thesecations are multi-valent, the possibility for multimetal-oligomer(s)ligand species and/or metal-multioligomer species were possible.However, scanning for these adducts at their predicted masses provedunsuccessful.

The results shown above for copper, iron, calcium, magnesium and zincmay be used as general teachings for subsequent analysis of the reactionbetween other metal ions and the inventive compounds, taking intoaccount such factors as oxidation state and the relative position in theperiodic table of the ion in question.

Example 36 MALDI—TOF/MS Analysis of High Molecular Weight ProcyanidinOligomers

An analytical examination was made on GPC eluants associated with highmolecular weight procyanidin oligomers as prepared in Example 3, MethodA. The objective was to determine whether procyanidin oligomers withn>12 were present. If present, these oligomers represent additionalcompounds of the invention. Adjustments to existing methods ofisolation, separation and purification embodied in the invention can bemade to obtain these oligomers for subsequent in vitro and in vivoevaluation for anti-cancer, anti-tumor or antineoplastic activity,antioxidant activity, inhibit DNA topoisomerase II enzyme, inhibitoxidative damage to DNA, and have antimicrobial, NO or NO-synthase,apoptosis, platelet aggregation, and blood or in vivo glucose-modulatingactivities, as well as efficacy as non-steroidal antiinflammatoryagents.

FIG. 59 represents a MALDI-TOF mass spectrum of the GPC eluant sampledescribed above. The [M+Na]⁺ and/or [M+K]⁺ and/or [M+2Na]⁺ ionscharacterizing procyanidin oligomers representative of tetramers throughoctadecamers are clearly evident.

It was learned that an acid and heat treatment will cause the hydrolysisof procyanidin oligomers. Therefore, the invention comprehends thecontrolled hydrolysis of high molecular weight procyanidin oligomers(e.g. where n is 13 to 18) as a method to prepare lower molecular weightprocyanidin oligomers (e.g. where n is 2 to 12).

Example 37 Dose Response Relationships of Procyanidin Oligomers andCanine and Feline Cell Lines

The dose respone effects of procyanidin oligomers were evaluated againstseveral canine and feline cell lines obtained from the Waltham Centerfor Pet Nutrition, Waltham on-the-Wolds, Melton Mowbray, Leicestershire,U.K. These cell lines were Canine normal kidney GH cell line;

Canine normal kidney MDCK cell line;

Feline normal kidney CRFK cell line; and

Feline lymphoblastoid FeA cell line producing leukemia virus which werecultured under the conditions described in Example 8, Method A.

Monomers and procyanidin oligomers, where n is 2 to 10 were purified asdescribed in Example 14, Method D. The oligomers were also examined byanalytical normal phase HPLC as described in Example 14, Method C, wherethe following results were obtained. Procyanidin % Purity by HPLCMonomers 95.4 Dimers 98.0 Trimers 92.6 Tetramers 92.6 Pentamers 93.2Hexamers 89.2 (Contains 4.4% pentamers) Heptamers 78.8 (Contains 18.0%hexamers) Octamers 76.3 (contains 16.4% heptamers) Nonamers 60.3(Contains 27.6% octamers) Decamers 39.8 (Contains 22.2% nonamers, 16.5%octamers, and 13.6% heptamers)

In those cases where the purity of the oligomer is <90%, methodsembodied in the invention are used for their repurification.

Each cell line was dosed with monomers and each procyanidin oligomer at10 ug/mL, 50 ug/mL and 100 ug/mL and the results shown in FIGS. 60-63.As shown in the Figures, high dose (100 ug/mL) administration ofindividual oligomers produced similar inhibitory effects on the felineFeA lymphoblastoid and feline normal kidney CRFK cell lines. In thesecases, cytotoxicity appeared with the tetramer, and increasingly higheroligomers elicited increasingly higher cytotoxic effects. By contrast,high dose (100 ug/mL) administration of individual oligomers to canineGH and MDCK normal kidney cell lines required a higher oligomer toinitiate the appearance of cytotoxicity. For the canine GH normal kidneycell line, cytotoxicity appeared with the pentamer. For the canine MDCKnormal kidney cell line, cytotoxicity appeared with the hexamer. In bothof these cases, the administration of higher oligomers producedincreasing levels of cytotoxicity.

Example 38 Tablet Formulations

A tablet formulation was prepared using cocoa solids obtained by methodsdescribed in U.S. application Ser. No. 08/709,406 filed 6 Sep. 1996 (nowU.S. Pat. No. 6,015,913), hereby incorporated herein by reference.Briefly, this edible material is prepared by a process which enhancesthe natural occurrence of the compounds of the invention in contrast totheir levels found in traditionally processed cocoa, such that the ratioof the initial amount of the compounds of the invention found in theunprocessed bean to that obtained after processing is less than or equalto 2. For simplicity, this cocoa solids material is designated herein asCP-cocoa solids. The inventive compound or compounds, e.g., in isolatedand/or purified form may be used in tablets as described in thisExample, instead of or in combination with CP-cocoa solids.

A tablet formula comprises the following (percentages expressed asweight percent): CP-cocoa solids 24.0% 4-Fold Natural vanilla extract(Bush Boake Allen) 1.5% Magnesium stearate (dry lubricant)(AerChem,Inc.) 0.5% Dipac tabletting sugar (Amstar Sugar Corp.) 37.0% Xylitol(American Xyrofin, Inc.) 37.0% 100.0%

The CP-cocoa solids and vanilla extract are blended together in a foodprocessor for 2 minutes. The sugars and magnesium stearate are gentlymixed together, followed by blending in the CP-cocoa solids/vanilla mix.This material is run through a Manesty Tablet Press (B3B) at maximumpressure and compaction to produce round tablets (15 mm×5 mm) weighing1.5-1.8 gram. Another tablet of the above mentioned formula was preparedwith a commercially available low fat natural cocoa powder (11% fat)instead of the CP-cocoa solids (11% fat). Both tablet formulas producedproducts having acceptable flavor characteristics and textureattributes.

An analysis of the two tablet formulas was performed using theprocedures described in Example 4, Method 2. In this case, the analysisfocused on the concentration of the pentamer and the total level ofmonomers and compounds of the invention where n is 2 to 12 which arereported below in Tab 15. TABLE 15 pentamer total pentamer total (ug/1.8g (ug/1.8 g Tablet sample (ug/g) (ug/g) serving) serving) tablet withCP-cocoa 239 8,277 430 14,989 solids tablet with ND 868 ND 1563commercial low fat cocoa powderND = not detected

The data clearly showed a higher level of pentamer and total level ofcompounds of the invention in the CP-cocoa solids tablet than in theother tablet formula. Thus, tablet formulas prepared with CP-cocoasolids are an ideal delivery vehicle for the oral administration ofcompounds of the invention, for pharmaceutical, supplement and foodapplications.

The skilled artisan in this area can readily prepare other tabletformulas covering a wide range of flavors, colors, excipients, vitamins,minerals, OTC medicaments, sugar fillers, UV protectants (e.g., titaniumdioxide, colorants, etc.), binders, hydrogels, and the like except forpolyvinyl pyrrolidone which would irreversibly bind the compounds of theinvention or combination of compounds. The amount of sugar fillers maybe adjusted to manipulate the dosages of the compounds of the inventionor combination of compounds.

Many apparent variations of the above are self-evident and possiblewithout departing from the spirit and scope of the example.

Example 39 Capsule Formulations

A variation of Example 38 for the oral delivery of the compounds of theinvention is made with push-fit capsules made of gelatin, as well assoft sealed capsules made of gelatin and a plasticizer such as glycerol.The push-fit capsules contain the compound of the invention orcombination of compounds or CP-cocoa solids as described in Examples 38and 40 in the form of a powder which can be optionally mixed withfillers such as lactose or sucrose to manipulate the dosages of thecompounds of the invention. In soft capsules, the compound of theinvention or combination of compounds or CP-cocoa solids are suspendedin a suitable liquid such as fatty oils or cocoa butter or combinationstherein. Since an inventive compound or compounds may belight-sensitive, e.g., sensitive to UV, a capsule can contain UVprotectants such as titanium dioxide or suitable colors to protectagainst UV. The capsules can also contain fillers such as thosementioned in the previous Example.

Many apparent variations of the above are self-evident and possible toone skilled in the art without departing from the spirit and scope ofthe example.

Example 40 Standard of Identity (SOI) and Non-Standard of Identity(non-SOI) Dark and Milk Chocolate Formulations

Formulations of the compounds of the invention or combination ofcompounds derived by methods embodied in the invention can be preparedinto SOI and non-SOI dark and milk chocolates as a delivery vehicle forhuman and veterinary applications. Reference is made to copending U.S.application Ser. No. 08/709,406, filed Sep. 6, 1996 (now U.S. Pat. No.6,015,913), hereby incorporated herein by reference. U.S. Ser. No.08/709,406 (now U.S. Pat. No. 6,015,913) relates to a method ofproducing cocoa butter and/or cocoa solids having conserved levels ofthe compounds of the invention from cocoa beans using a uniquecombination of processing steps. Briefly, the edible cocoa solidsobtained by this process conserves the natural occurrence of thecompounds of the invention in contrast to their levels found intraditionally processed cocoa, such that the ratio of the initial amountof the compounds of the invention found in the unprocessed bean to thatobtained after processing is less than or equal to 2. For simplicity,this cocoa solids material is designated herein as CP-cocoa solids. TheCP-cocoa solids are used as a powder or liquor to prepare SOI andnon-SOI chocolates, beverages, snacks, baked goods, and as an ingredientfor culinary applications.

The term “SOI chocolate” as used herein shall mean any chocolate used infood in the United States that is subject to a Standard of Identityestablished by the U.S. Food and Drug Administration under the FederalFood, Drug and Cosmetic Act. The U.S. definitions and standards forvarious types of chocolate are well established. The term “non-SOIchocolate” as used herein shall mean any nonstandardized chocolateswhich have compositions which fall outside the specified ranges of thestandardized chocolates.

Examples of nonstandardized chocolates result when the cocoa butter ormilk fat are replaced partially or completely; or when the nutrativecarbohydrate sweetener is replaced partially or completely; or flavorsimitating milk, butter, cocoa powder, or chocolate are added or otheradditions or deletions in the formula are made outside the U.S. FDAStandards of Identity for chocolate or combinations thereof.

As a confection, chocolate can take the form of solid pieces ofchocolate, such as bars or novelty shapes, and can also be incorporatedas a component of other, more complex confections where chocolate isoptionally combined with any Flavor & Extract Manufacturers Association(FEMA) material, natural juices, spices, herbs and extracts categorizedas natural-flavoring substances; nature-identical substances; andartificial flavoring substances as defined by FEMA GRAS lists, FEMA andFDA lists, Council of Europe (CoE) lists, International Organization ofthe Flavor Industry (IOFI) adopted by the FAO/WHO Food StandardProgramme, Codex Alimentarius, and Food Chemicals Codex and generallycoats other foods such as caramel, nougat, fruit pieces, nuts, wafers orthe like. These foods are characterized as microbiologicallyshelf-stable at 65-85° F. under normal atmospheric conditions. Othercomplex confections result from surrounding with chocolate softinclusions such as cordial cherries or peanut butter. Other complexconfections result from coating ice cream or other frozen orrefrigerated desserts with chocolate. Generally, chocolate used to coator surround foods must be more fluid than chocolates used for plainchocolate solid bars or novelty shapes.

Additionally, chocolate can also be a low fat chocolate comprising a fatand nonfat solids, having nutrative carbohydrate sweetener(s), and anedible emulsifier. As to low fat chocolate, reference is made to U.S.Pat. Nos. 4,810,516,4,701,337, 5,464,649, 5,474,795, and WO 96/19923.

Dark chocolates derive their dark color from the amount of chocolateliquor, or alkalized liquor or cocoa solids or alkalized cocoa solidsused in any given formulation. However, the use of alkalized cocoasolids or liquor would not be used in the dark chocolate formulations inthe invention, since Example 27, Table 13 teaches the loss of thecompounds of the invention due to the alkalization process.

Examples of formulations of SOI and non-SOI dark and milk chocolates arelisted in Tables 16 and 17. In these formulations, the amount of thecompounds of the invention present in CP-cocoa solids was compared tothe compounds of the invention present in commercially available cocoasolids.

The following describes the processing steps used in preparing thesechocolate formulations.

Process for Non-SOI Dark Chocolate

-   1. Keep all mixers and refiners covered throughout process to avoid    light.-   2. Batch all the ingredients excluding 40% of the free fat (cocoa    butter and anhy. milk fat) maintaining temperature between 30-35° C.-   3. Refine to 20 microns.-   4. Dry conche for 1 hour at 35° C.-   5. Add full lechithin and 10% cocoa butter at the beginning of the    wet conche cycle; wet conche for 1 hour.-   6. Add all remaining fat, standardize if necessary and mix for 1    hour at 3°5C.-   7. Temper, mould and package chocolate.    Process for SOI Dark Chocolate-   1. Batch all ingredients excluding milk fat at a temperature of 60°    C.-   2. Refine to 20 microns.-   3. Dry conche for 3.5 hours at 60° C.-   4. Add lecithin and milk fat and wet conche for 1 hour at 60° C.-   5. Standardize if necessary and mix for 1 hour at 35° C.    Temper, mould and package chocolate.    Process for Non-SOI Milk Chocolate-   1. Keep all mixers and refiners covered throughout process to avoid    light.-   2. Batch sugar, whole milk powder, malted milk powder, and 66% of    the cocoa butter, conche for 2 hours at 75° C.-   3. Cool batch to 35° C. and add cocoa powder, ethyl vanillin,    chocolate liquor and 21% of cocoa butter, mix 20 minutes at 35° C.-   4. Refine to 20 microns.-   5. Add remainder of cocoa butter, dry conche for 1.5 hour at 35° C.-   6. Add anhy. milk fat and lecithin, wet conche for 1 hour at 35° C.-   7. Standardize, temper, mould and package the chocolate.    Process for SOI Milk Chocolate-   1. Batch all ingredients excluding 65% of cocoa butter and milk fat    at a temperature of 60° C.-   2. Refine to 20 microns.-   3. Dry conche for 3.5 hours at 60° C.-   4. Add lecithin, 10% of cocoa butter and anhy. milk fat; wet conche    for 1 hour at 60° C.-   5. Add remaining cocoa butter, standardize if necessary and mix for    1 hour at 35° C.-   6. Temper, mould and package the chocolate.

The CP-cocoa solids and commercial chocolate liquors used in theformulations were analyzed for the pentamer and total level of monomersand compounds of the invention where n is 2 to 12 as described in Method2, Example 4 prior to incorporation in the formulations. These valueswere then used to calculate the expected levels in each chocolateformula as shown in Tables 16 and 17. In the cases for the non-SOI darkchocolate and non-SOI milk chocolate, their products were similarlyanalyzed for the pentamer, and the total level of monomers and thecompounds of the invention where n is 2 to 12. The results appear inTables 16 and 17.

The results from these formulation examples indicated that SOI andnon-SOI dark and milk chocolates formulated with CP-cocoa solidscontained approximately 6.5 times more expected pentamer, and 3.5 timesmore expected total levels in the SOI and non-SOI dark chocolates; andapproximately 4.5; 7.0 times more expected pentamer and 2.5; 3.5 timesmore expected total levels in the SOI and non-SOI milk chocolates,respectively.

Analyses of some of the chocolate products were not performed since thedifference between the expected levels of the compounds of the inventionpresent in finished chocolates prepared with CP-cocoa solids weredramatically higher than those formulas prepared with commerciallyavailable cocoa solids. However, the effects of processing was evaluatedin the non-SOI dark and milk chocolate products. As shown in the tables,a 25-50% loss of the pentamer occurred, while slight differences intotal levels were observed. Without wishing to be bound by any theory,it is believed that these losses are due to heat and/or low chain fattyacids from the milk ingredient (e.g. acetic acid, propionic acid andbutyric acid) which can hydrolyze the oligomers (e.g. a trimer canhydrolyze to a monomer and dimer). Alternatively, time consumingprocessing steps can allow for oxidation or irreversible binding of thecompounds of the invention to protein sources within the formula. Thus,the invention comprehends altering methods of chocolate formulation andprocessing to address these effects to prevent or minimize these losses.

The skilled artisan will recognize many variations in these examples tocover a wide range of formulas, ingredients, processing, and mixtures torationally adjust the naturally occurring levels of the compounds of theinvention for a variety of chocolate applications. TABLE 16 DarkChocolate Formulas Prepared with non-Alkalized Cocoa Ingredients SOIDark Non-SOI Dark SOI Dark Chocolate Chocolate Chocolate UsingCommercial Using CP-cocoa solids Using CP-Cocoa Solids Cocoa SolidsFormulation: Formulation: Formulation: 41.49% Sugar 41.49% sugar 41.49%sugar    3% whole milk    3% whole milk    3% whole milk powder powderpowder   26% CP-cocoa 52.65% CP-liquor 52.65% com. liquor solids  2.35%anhy. milk fat  2.35% anhy. milk fat  4.5% com. liquor  0.01% vanillin 0.01% vanillin 21.75% cocoa butter  0.5% lecithin  0.5% lecithin  2.75%anhy. milk fat  0.01% vanillin  0.5% lecithin Total fat: 31% Total fat:31% Total fat: 31% Particle size: Particle size: Particle size: 20microns 20 microns 20 microns Expected Levels of pentamer and totaloligomeric procyanidins (monomers and n = 2-12; units of ug/g) Pentamer:1205 Pentamer: 1300 Pentamer: 185 Total: 13748 Total: 14646 Total: 3948Actual Levels of pentamer and total oligomeric procyandins (monomers andn = 2-12; units of ug/g) Pentamer: 561 Not performed Not performedTotal: 14097

TABLE 17 Milk Chocolate Formulas Prepared with non-Alkalized CocoaIngredients Non-SOI Milk Chocolate SOI Milk Chocolate Using SOI MilkChocolate Using Using CP-cocoa solids CP-Cocoa Solids Commercial CocoaSolids Formulation: Formulation: Formulation: 46.9965% Sugar 46.9965%sugar 46.9965% sugar   15.5% whole milk powder   15.5% whole milk powder  15.5% whole milk powder   4.5% CP-cocoa solids   13.9% CP-liquor  13.9% com. liquor   5.5% com. liquor   1.6% anhy. milk fat   1.60%anhy. milk fat   21.4% cocoa butter  0.0035% vanillin  0.0035% vanillin  1.6% anhy. milk fat   0.5% lecithin   0.5% lecithin  0.035% vanillin  17.5% cocoa butter   17.5% cocoa butter   0.5% lecithin   4.0% maltedmilk powder   4.0% malted milk powder   4.0% malted milk powder Totalfat: 31.75% Total fat: 31.75% Total fat: 31.75% Particle size: 20microns Particle size: 20 microns Particle size: 20 microns ExpectedLevels of pentamer and total oligomeric procyanidins (monomers and n =2-12; units of ug/g) Pentamer: 225 Pentamer: 343 Pentamer: 49 Total:2734 Total: 3867 Total: 1042 Actual Levels of pentamer and totaloligomeric procyandins (monomers and n = 2-12; units of ug/g) Pentamer:163 Not performed Not performed Total: 2399

Example 41 Hydrolysis of Procyanidin Oligomers

Example 14, Method D describes the preparation normal phase HPLCprocedure to purify the compounds of the invention. The oligomers areobtained as fractions dissolved in mobile phase. Solvent is then removedby standard vacuum distillation (20-29 in. Hg: 40° C.) on a Rotovapapparatus. It was observed that losses of a particular oligomer occurredwith increases in smaller oligomers when the vacuum distillationresidence time was prolonged or temperatures >40° C. were used.

The losses of a particular oligomer with accompanying increases insmaller oligomers was attributed to a time-temperature acid hydrolysisfrom residual acetic acid present in the mobile phase solvent mixture.This observation was confirmed by the following experiment where 100 mgof hexamer was dissolved in 50 mL of the mobile phase containingmethylene chloride, acetic acid, water, and methanol (see Example 14,Method D for solvent proportions) and subjected to a time-temperaturedependent distillation. At specific times, an aliquot was removed foranalytical normal phase HPLC analysis as described in Example 4, Method2. The results are illustrated in FIGS. 64 and 65, where hexamer levelsdecreased in a time-temperature dependent fashion. FIG. 65 illustratesthe appearance of one of the hydrolysis products (Trimer) in atime-temperature dependent fashion. Monomer and other oligomers (dimerto pentamer) also appeared in a time-temperature dependent fashion.

These results indicated that extreme care and caution must be takenduring the handling of the inventive polymeric compounds.

The results provided above, together with that found in Examples 5, 15,18, 19, 20 and 29, demonstrate that the method described above can beused to complement other methods embodied in the invention to identifyany given oligomer of the invention.

For instance, the complete hydrolysis of any given oligomer which yieldsexclusively (+)-catechin or (−)-epicatechin eliminates many “mixed”monomer-based oligomer structure possibilities and reduces thestereochemical linkage possibilities characteristic for each monomercomprising any given oligomer.

Further, the complete hydrolysis of any given oligomer which yields both(+)-catechin and (−)-epicatechin in specific proportions provides theskilled artisan with information on the monomer composition of any givenoligomer, and hence, the stereochemical linkage possibilitiescharacteristic for each monomer comprising the oligomer.

The skilled artisan would recognize the fact that acid catalyzedepimerization of individual monomers can occur and suitable controlexperiments and nonvigorous hydrolysis conditions should be taken intoaccount (e.g., the use of an organic acids, such as acetic acid, in lieuof concentrated HCl, HNO₃, etc).

Having thus described in detail the preferred embodiments of the presentinvention, it is to be understood that the invention defined by theappended claims is not to be limited by particular details set forth inthe above descriptions as many apparent variations thereof are possiblewithout departing from the spirit or scope of the present invention.

REFERENCES

-   1. Barrows, L. R., Borchers, A. H., and Paxton, M. B., Transfectant    CHO Cells Expressing 0⁶-alkylguanine—DNA-alkyltransferase Display    Increased Resistance to DNA Damage Other than O⁶-guanine Alkylation,    Carcinogenesis, 8:1853 (1987).-   2. Boukharta, M., Jalbert, G. and Castonguay, A., Efficacy of    Ellagitannins and Ellagic Acid as Cancer Chemopreventive    Agents—Presented at the XVI^(th) International Conference of the    Groupe Polyphenols, Lisbon, Portugal, Jul. 13-16, 1992.-   3. Burres, N. S., Sazesh, J., Gunawardana, G. P., and Clement, J.    J., Antitumor Activity and Nucleic Acid Binding Properties of    Dercitin, a New Acridine Alkaloid Isolated from a Marine Dercitus    species Sponge, Cancer Research, 49, 5267-5274 (1989).-   4. Caragay, A. B., Cancer Preventive Foods and Ingredients, Food    Technology, 46:4, 65-79 (1992).-   5. Chu, S.-C., Hsieh, Y.-S. and Lim, J.-Y., Inhibitory Effects of    Flavonoids on Maloney Murine Leukemia Virus Reverse Transcriptase    Activity, J. of Natural Products, 55:2, 179-183 (1992).-   6. Clapperton, J., Hammerstone, J. F. Jr., Romanczyk, L. J. Jr.,    Chan, J., Yow, S., Lim, D. and Lockwood, R., Polyphenols and Cocoa    Flavor—Presented at the XVI^(th) International Conference of the    Groupe Polyphenols, Lisbon, Portugal, Jul. 13-16, 1992.-   7. Danks, M. K., Schmidt, C. A., Cirtain, M. C., Suttle, D. P., and    Beck, W. T., Altered Catalytic Activity of and DNA Cleavage by DNA    Topoisomerase II from Human Leukemic Cells Selected for Resistance    to VM-26, Biochem., 27:8861 (1988).-   8. Delcour, J. A., Ferreira, D. and Roux, D. G., Synthesis of    Condensed Tannins, Part 9, The Condensation Sequence of    Leucocyanidin with (+)-Catechin and with the Resultant    Procyanidins, J. Chem. Soc. Perkin Trans. I, 1711-1717 (1983).-   9. Deschner, E. E., Ruperto, J., Wong, G. and Newmark, H. L.,    Quercetin and Rutin as Inhibitors of Azoxymethanol—Induced Colonic    Neoplasia, Carcinogenesis, 7, 1193-1196 (1991).-   10. Designing Foods, Manipulating Foods to Promote Health, Inform,    4:4, 344-369 (1993).-   11. Drake, F. H., Hofmann, G. A., Mong., S.-M., Bartus, J. O.,    Hertzberg, R. P., Johnson, R. K., Mattern, M. R., and Mirabelli, C.    K., in vitro and Intercellular Inhibition of Topoisomerase II by the    Antitumor Agent Membranone, Cancer Research, 49, 2578-2583 (1989).-   12. Engels J. M. M., Genetic Resources of Cacao: A Catalogue of the    CATIE Collection, Tech. Bull. 7, Turrialba, Costa Rica (1981).-   13. Enriquez G. A. and Soria J. V., Cocoa Cultivars Register IICA,    Turrialba, Cost Rica (1967).-   14. Ferreira, D., Steynberg, J. P., Roux, D. G. and Brandt, E. V.,    Diversity of Structure and Function in Oligomeric Flavanoids,    Tetrahedron, 48:10, 1743-1803 (1992).-   15. Fesen, M. and Pommier, Y., Mammalian Topoisomerase II Activity    is Modulated by the DNA Minor Groove Binder—Distainycin in Simian    Virus 40 DNA, J. Biol. Chem., 264, 11354-11359 (1989).-   16. Fry, D. W., Boritzki, T. J., Besserer, J. A., and Jackson, R.    C., in vitro Strand Scission and Inhibition of Nucleic Acid    Synthesis on L1210 Leukemia Cells by a New Class of DNA Complexes,    the anthra [1, 9-CD]pyrazol-6(2H)-ones (anthrapyrazoles), Biochem.    Pharmacol., 34, 3499-3508 (1985).-   17. Hsiang, Y.-H., Jiang, J. B., and Liu, L. F., Topoisomerase II    Mediated DNA Cleavage by Amonafide and Its Structural Analogs, Mol.    Pharmacol., 36, 371-376 (1989).-   18. Jalal, M. A. F. and Collin, H. A., Polyphenols of Mature Plant,    Seedling and Tissue Cultures of Theobroma Cacoa, Phytochemistry, 6,    1377-1380 (1978).-   19. Jeggo, P. A., Caldecott, K., Pidsley, S., and Banks, G. R.,    Sensitivity of Chinese Hamster Ovary Mutants Defective in DNA Double    Strand Break Repair to Topoisomerase II Inhibitors, Cancer Res.,    49:7057 (1989).-   20. Kashiwada, Y., Nonaka, G.-I., Nishioka, I., Lee, K. J.-H., Bori,    I., Fukushima, Y., Bastow, K. F., and Lee, K.-H., Tannin as Potent    Inhibitors of DNA Topoisomerase II in vitro, J. Pharm. Sci., 82:5,    487-492 (1993).-   21. Kato, R., Nakadate, T., Yamamoto, S. and Sugimura, T.,    Inhibition of 12-O-tetradecanoylphorbol-13-acetate Induced Tumor    Promotion and Ornithine Decarboxylase Activity by Quercitin:    Possible Involvement of lipoxygenase Inhibition, Carcinogenesis, 4,    1301-1305 (1983).-   22. Kawada, S.-Z., Yamashita, Y., Fujii, N. and Nakano, H.,    Induction of Heat Stable Topoisomerase II-DNA Cleavable Complex by    Nonintercalative Terpenoids, Terpentecin and Clerocidin, Cancer    Research, 51, 2922-2929 (1991).-   23. Kemp, L. M., Sedgwick, S. G. and Jeggo, P. A., X-ray Sensitive    Mutants of Chinese Hamster Ovary Cells Defective in Double Strand    Break Rejoining, Mutat. Res., 132:189 (1984).-   24. Kikkoman Corporation, Antimutagenic Agent Containing    Proanthocyanidin Oligomer Preferably Having Flavan-3-ol-Diol    Structure, JP 04190774-A, Jul. 7, 1992.-   25. Lehrian, D. W.; Patterson, G. R. In Biotechnology; Reed, G.,    Ed.; Verlag Chemie: Weinheim, 1983, Vol. 5, Chapter 12.-   26. Leonessa, F., Jacobson, M., Boyle, B., Lippman, J., McGarvey,    M., and Clarke, R. Effect of Tamoxifen on the Multidrug-Resistant    Phenotype in Human Breast Cancer Cells: Isobolograms, Drug    Accumulation, and Mr 170,000 Glycoprotein (gp 170) Binding Studies,    Cancer Research, 54, 441-447 (1994).-   27. Liu, L. F., DNA Toposimerase Poisons as Antitumor Drugs, Ann.    Rev. Biochem., 58, 351-375 (1989).-   28. McCord, J. D. and Kilara A. Control of Enzymatic Browning in    Processed Mushrooms (Agaricus bisporus). J. Food Sci., 48:1479    (1983).-   29. Miller, K. G., Liu, L. F. and Englund, P. A., Homogeneous Type    II DNA Topoisomerase from Hela Cell Nuclei, J. Biol. Chem., 256:9334    (1981).-   30. Mosmann, T., Rapid Colorimetric Assay for Cellular Growth and    Survival: Application to Proliferation and Cytoxicity Assays, J.    Immunol. Methods, 65, 55 (1983).-   31. Muller, M. T., Helal, K., Soisson, S. and Spitzer, J. R., A    Rapid and Quantitative Microtiter Assay for Eukaryotic Topoisomerase    II, Nuc. Acid Res., 17:9499 (1989).-   32. Nawata, H., Chong, M. T., Bronzert, D. and Lippman, M. E.    Estradiol-Independent growth of a Subline of MCF-7 Human Breast    Cancer Cells in Culture, J. Biol. Chem., 256:13, 6895-6902 (1981).-   33. Okuda, T., Yoshida, T., and Hatano, T., Molecular Structures and    Pharmacological Activities of Polyphenols—Oligomeric Hydrolyzable    Tannins and Others—Presented at the XVI^(th) International    Conference of the Groupe Polyphenols, Lisbon, Portugal, Jul. 13-16,    1992.-   34. Phenolic Compounds in Foods and Their Effects on Health II.    Antioxidants & Cancer Prevention, Huang, M.-T., Ho, C.-T., and    Lee, C. Y. editors, ACS Symposium Series 507, American Chemical    Society, Washington, D.C. (1992).-   35. Phenolic Compounds in Foods and Their Effects on Health I,    Analysis, Occurrence & Chemistry, Ho, C.-T., Lee, C. Y., and Huang,    M.-T editors, ACS Symposium Series 506, American Chemical Society,    Washington, D.C. (1992).-   36. Porter, L. J., Ma, Z. and Chan, B. G., Cocoa Procyanidins: Major    Flavanoids and Identification of Some Minor Metabolites,    Phytochemistry, 30, 1657-1663 (1991).-   37. Revilla, E., Bourzeix, M. and Alonso, E., Analysis of Catechins    and Procyanidins in Grape Seeds by HPLC with Photodiode Array    Detection, Chromatographia, 31, 465-468 (1991).-   38. Scudiero, D. A., Shoemaker, R. H., Paull, K. D., Monks, A.,    Tierney, S., Nofziger, T. H., Currens, M. J., Seniff, D., and    Boyd, M. R. Evaluation of a Soluble Tetrazolium/Formazan Assay for    Cell Growth and Drug Sensitivity in Culture Using Human and Other    Tumor Cell Lines, Canur Research, 48, 4827-4833 (1988).-   39. Self, R., Eagles, J., Galletti, G. C., Mueller-Harvey, I.,    Hartley, R. D., Lee, A. G. H., Magnolato, D., Richli, U., Gujur, R.    and Haslam, E., Fast Atom Bombardment Mass Spectrometry of    Polyphenols (syn. Vegetable Tannins), Biomed Environ. Mass Spec. 13,    449-468 (1986).-   40. Tanabe, K., Ikegami, Y., Ishda, R. and Andoh, T., Inhibition of    Topoisomerase II by Antitumor Agents bis(2,6-dioxopiperazine)    Derivatives, Cancer Research, 51, 4903-4908 (1991).-   41. Van Oosten, C. W., Poot, C. and A. C. Hensen, The Precision of    the Swift Stability Test, Fette, Seifen, Anstrichmittel, 83:4,    133-135 (1981).-   42. Wang, J. C., DNA Topoisomerases, Ann. Rev. Biochem., 54, 665-697    (1985).-   43. Warters, R. L., Lyons, B. W., Li, T. M. and Chen, D. J.,    Topoisomerase II Activity in a DNA Double-Strand Break Repair    Deficient Chinese Hamster Ovary Cell Line, Mutat. Res., 254:167    (1991).-   44. Yamashita, Y., Kawada, S.-Z. and Nakano, H., Induction of    Mammalian Topoismerase II Dependent DNA Cleavage by Nonintercalative    Flavanoids, Genistein and Orbol., Biochem Pharm, 39:4, 737-744    (1990).-   45. Yamashita, Y., Kawada, S.-Z., Fujii, N. and Nakano, H.,    Induction of Mammalian DNA Topoisomerase I and II Mediated DNA    Cleavage by Saintopin, a New Antitumor Agent from Fungus, Biochem.,    30, 5838-5845 (1991).-   46. Feldman, P. L., Griffith, O. W., and Stuehr, D. J. The    Surprising Life of Nitric Oxide, Chem. & Eng. News, Dec. 20,    1993, p. 26-38.-   47. Jia, L., Bonaventura, C. and Stamler, J. S.,    S-Nitrosohaemoglobin: A Dynamic Activity of Blood Involved in    Vascular Control, Nature, 380, 221-226 (1996).-   48. Radomski, M. W., Palmer, R. M. J. and Moncada, S. Comparative    Pharmacology of Endothelium Derived Relaxing Factor, Nitric Oxide    and Prostacyclin in Platelets, Brit. J. Pharmacol, 92, 789-795    (1989).-   49. Stamler, J. S., Mendelshon, M. E., Amarante, P., Smick, D.,    Andon, N., Davies, P. F., Cooke, J. P., and Loscalzo,    N-Acetylcysteine Potentiates platelet Inhibitio By    Edothelium—Derived Relaxing Factor, J. Circ. Research, 65, 789-795    (1989).-   50. Bath, P. M. W., Hassall, D. G., Gladwin, A. M., Palmer, R. M. J.    and Martin, J. F., Nitric Oxide and Prostacyclin. Divergence of    Inhibitory Effects on Monocyte Chemotaxis and Adhesion to    endothelium In Vitro. Arterioscl. Throm., 11, 254-260 (1991).-   51. Garg, U. C. and Hassid, A. Nitric Oxide Generating Vasodilators    and 8-Bromo-Cyclicguanosine Monophosphate Inhibit Mitogenesis and    Proliferation of Cultured Rat Vascular Smoothe Muscle Cells, J.    Clin. Invest., 83, 1774-1777 (1989).-   52. Creager, M. A., Cooke, J. P., Mendelsohn, M. E., Gallagher, S.    J., Coleman, S. M., Loscalzo, J. and Dzau, V. J. Impaired    Vasodilation of Forearm Resistance Vessels in Hypercholesterolemic    Humans, J. Clin. Invest., 86, 228-234 (1990).-   53. Steinberg, D., Parthasarathy, S., Carew, T. E., Khoo, J. C. and    Witztum, J. L. Beyond Cholesterol. Modifications of Low Density    Lipoproteins that Increase its Atherogenicity. The New England J. of    Med, 320, 915-924 (1989).-   54. Tsuiji, M. and DuBois, R. N. Alterations in Cellular Adhesion    and Apoptosis in Epithelial Cells Overexpressing Prostaglandin    Endoperoxide Synthase 2, Cell, 83, 493-501 (1995).-   55. Marcus, A. J. Aspirin as Prophylaxis Colorectal Cancer, The New    Eng. J. Med., 333: 10, 656-658 (1995).-   56. P. J. Pastricha, Bedi, A., O'Connor, K., Rashid, A., Akhatar, A.    J., Zahurak, M. L., Piantadosi, S., Hamilton, S. R. and    Giardiello, F. M. The Effects of Sulindac on Colorectal    Proliferation and Apoptosis in Familial Adenomatous Polyopsis.    Gastroenterology, 109, 994-998 (1995).-   57. Lu, X., Xie, W., Reed, D., Bradshaw, W. and Simmons, D.    Nonsteroidal Anti Inflammatory Drugs Cause Apoptosis and Induce    Cyclooxygenase in Chicken Embryo Fibroblasts. P.N.A.S. U.S.A., 92,    7961-7965 (1995).-   58. Gajewski, T. F. and Thompson, C. B. Apoptosis Meets Signal    Transduction: Elimination of A BAD Influence. Cell, 87, 589-592    (1996).-   59. Funk, C. D., Funk, L. B., Kennedy, M. E., Pong, A. S. and    Fitzgerald, G. A. Human platelet/Erythroleukemiz Cell Prostaglandin    G/H Synthase: cDNA Cloning, Expression and Gene Chromosomal    Assignment, FASEB J., 5: 2304-2312 (1991).-   60. Patrono, C. Aspirin as an Antiplatelet Drug. The New Eng. J.    Med., 333: 18, 1287-1294 (1994).-   61. Howell, T. H. and Williams, R. C. Nonsteroidal Antiinflammatory    Drugs as Inhibitors of Periodonal Disease Progression. Crit. Rev. of    Oral Biol & Med., 4: 2, 117-195 (1993).-   62. Brisham, M. B. Oxidants and Free Radicals in Inflammatory Bowel    Disease. Lancet, 344, 859-861 (1994).-   63. Oates, J. A. The 1982 Nobel Prize in Physiology and Medicine,    Science, 218, 765-768 (1996).-   64. Hunter, T. and Pines, J. Cyclins and Cancer II: Cyclin D and CDK    Inhibitors Come of Age, Cell, 79, 573-582 (1994).-   65. King, R. W., Jackson, P. K. and Kirschner, M. W. Mitosis in    Transition, Cell, 79, 563-571 (1994).-   66. Sherr, C. J. G1 Phase Progession: Cycling on Cue, Cell, 79,    551-555 (1994).-   67. Nurse, P. Ordering S Phase and M Phase in the Cell Cycle, Cell,    79, 547-550 (1994).-   68. DeCross, A. J., Marshall, B. J., McCallum, R. W., Hoffman, S.    R., Barrett, L. J. and Guerrant, R. L. Metronidazole Susceptibility    Testing for Helicobacter pylori: Comparison of Disk, Broth and Agar    Dilution Methods and Their Clinical Relevance, J. Clin. Microbiol.,    31, 1971-1974 (1993).-   69. Anon., Flavor and Fragrance Materials—1981: Worldwide reference    list of materials used in compounding flavors and fragrances,    Chemical Sources Association, Allured Publishing Corp.-   70. van Rensburg, H., van Heerden, P. S., Bezuidenhoudt, B. C. B.    and Ferreira, D., The first enantioselective synthesis of trans- and    cis-dihydroflavanols, Chem. Comm. 24, 2705-2706 (1996).-   71. Lockhart, D. J., Dong, H., Byrne, M. C., Follettre, M. T.,    Gallo, M. V., Chee, M. S., Mittmann, M., Wang, C., Kobayashi, M.,    Horton, H., Brown, E. L. Expression Monitoring by Hybridization to    High-Density Oligonucleotide Assays, Nature Biotechnology, 14,    1675-1680 (1996).-   72. Winyard, P. G. and Blake, D. R. Antioxidants, Redox-Regulated    Transcription Factors, and Inflammation, Advances in Pharmacology,    38, 403-421 (1997).-   73. Schwartz, M. A., Rose, B. F., Holton, R. A., Scott, S. W. and    Vishnuvajjala, B. Intramolecular Oxidative Coupling of Diphenolic,    Monophenolic and Nonphenolic Substrates, J. Am. Chem. Soc. 99: 8,    2571-2575 (1977).-   74. Greene, T. W. “Protecting Groups in Organic Synthesis”, Wiley,    New York (1981).-   75. Warren, S. “Designing Organic Syntheses. A Programmed    Introduction to the Synthon Approach”, Wiley, New York (1978).-   76. Collman, J. P., Hegedus, L. S., Norton, J. R. and Finke, R. G.    “Principles and Applications of Organotransition Metal Chemistry”,    University Science Books (1987).-   77. Tsujii, M. and DuBois, R. N. Alterations in Cellular Adhesion    and Apoptosis in Epithelial Cells Overexpressing Prostaglandin    Endoperoxide Synthase 2, Cell, 83, 493-501 (1995).-   78. Pashricha, P. J., Bedi, A., O'Connor, K., Rashid, A., Akhtar, D.    J., Zahurak, M. L., Piantadosi, S., Hamilton, S. R. and    Giardiello, F. M. The Effects of Sulindac on Colorectal    Proliferation and Apoptosis in Familial Adenomatous Polyposis,    Gastroenterology, 109, 994-998 (1995).-   79. Verhagan, J. V., Haenen, G. R. M. M. and Bast, A. Nitric Oxide    Radical Scavenging by Wines, J. Agric. Food Chem. 44, 3733-3734    (1996).-   80. Aruoma, O. I. Assessment of Potential Prooxidant and Antioxidant    Actions, J.A.O.C.S., 73: 12, 1617-1625.-   81. Stoner, G. D. and Mukhtar, H. Polyphenols as Cancer    Chemopreventive Agents, J. Cell. Biochem. 22, 169-180 (1995).-   82. Gali, H. U., Perchellet, E. M., Klish, D. S., Johnson, J. M. and    Perchellet, J-P. Antitumor-promoting activities of hydrolyzable    tannins in mouse skin, Carcinogenesis, 13: 4, 715-718 (1992).-   83. Tabib, K., Besancon, P. and Rouanet, J-M. Dietary Grape Seed    Tannins Affect Lipoproteins, Lipoprotein Lipases and Tissue Lipids    in Rats Fed Hypercholesterolemic Diets, J. Nutrition, 124:12,    2451-2457.-   84. Paolino, V. J. and Kashket, S. Inhibition by Cocoa Extracts of    Biosynthesis of Extracellular Polysaccharide by Human Oral Bacteria,    Archs. Oral Biol. 30:4, 359-363 (1985).-   85. Lockhart, D. J., Dong, H., Byrne, M. C., Follettie, M. T.,    Gallo, M. V., Chee, M. S., Mittmann, M., Wang, C., Kobayashi, M.,    Horton, H., and Brown, E. L., Expression monitoring by hybridization    to high-density oligonucleotide arrays, Nature Biotech., 14,    1675-1680 (1996).-   86. Kreiner, T. Rapid genetic sequence analysis using a DNA probe    array system, Am. Lab., March, 1996.-   87. Lipshutz, R. J., Morris, D., Chee, M., Hubbell, E., Kozal, M.    J., Shah, N., Shen, N., Yang, R. and Fodor, S. P. A. Using    Oligonucleotide Probe Arrays to Access Genetic Diversity,    Biotechniques, 19: 3, 442-447 (1995).-   88. Borman, S. DNA Chips Come of Age, Chem. & Eng. News, 42-43, Dec.    9, 1996-   89. Tahara, H., Mihara, Y., Ishii, Y., Fujiwara, M., Endo, H.,    Maeda, S and Ide, T. Telomerase Activity in Cellular    Immortalization, Cell Structure and Function, 20: 6, IB-1615 (1995).-   90. Heller, K., Kilian, A., Paityszek, M. A., and Kleinhofs, A.    Telomerase activity in plant extracts, Mol. Gen. Genet. 252, 342-345    (1996).-   91. Goffeau, A. Molecular fish on chips, Nature, 385, 202-203    (1997).-   92. Friedrich, G. A. Moving beyond the genome projects, Nature    Biotechnology, 14, 1234-1237 (1996).-   93. Blanchard, R. K. and Cousins, R. J. Differential display of    intestingal mRNAs regulated by dietary zinc., Proc. Natl. Acad. Sci.    USA, 93, 6863-6868 (1996).-   94. Pennisi, E. Opening the Way to Gene Activity, Science, 275:    155-157 (1997).-   95. Medlin, J. The Amazing Shrinking Laboratory, Environmental Healt    Perspectives, 103: 3, 244-246.-   96. Luehrsen, K. R., Marr, L. L., van der Knaap, E. and    Cumberledge, S. Analysis of Differential Display RT-PCR Products    Using Fluorescent Primers and GENESCAN Software, Biotechniques, 22:    1, 168-174.-   97. Geiss, F., Heinrich, M., Hunkler, D. and Rimpler, H.    Proanthocyanidins with (+)-Epicatechin Units from Byronima    Crassifolia Bark, Phytochemistry, 39: 1, 635-643 (1995).-   98. Iibuchi, S., Minoda, Y. and Yamada, K. Studies on Tannin Acyl    Hydrolase of Microorganisms, Part II. A New Method Determining the    Enzyme Activity Using the Change of Ultra Violet Absorption, Agr.    Biol. Chem. 31: 5, 513-518 (1967).-   99. Ferreira, D., Steynberg, J. P., Roux, D. G. and Brandt, E. V.    Diversity of Structure and Function in Oligomeric Flavanoids,    Tetrahedron, 48: 10, 1743-1803 (1992).

1. A polymeric compound of the formula A_(n), wherein A is a monomer ofthe formula:

wherein n is an integer from 3 to 18, such that there is at least oneterminal monomeric unit A, and a plurality of additional monomericunits; R is 3-(α)-OH, 3-(β)-OH, 3-(α)-O-sugar, or 3-(β)-O-sugar; bondingbetween adjacent monomers takes place at positions selected from thegroup consisting of 4, 6 and 8; a bond for an additional monomeric unitin position 4 has alpha or beta stereochemistry; X, Y and Z are selectedfrom the group consisting of monomeric unit A, hydrogen, and a sugar,with the provisos that as to the at least one terminal monomeric unit,bonding of the additional monomeric unit thereto is at position 4 andoptionally Y=Z=hydrogen; the sugar is optionally substituted with aphenolic moiety, and pharmaceutically acceptable salts, derivativesthereof, and oxidation products thereof.
 2. The compound of claim 1wherein n is
 5. 3. The compound of claim 1 wherein the sugar is selectedfrom the group consisting of glucose, galactose, xylose, rhamnose andarabinose.
 4. The compound of claim 1 which is isolated from a naturalsource.
 5. The compound of claim 4 wherein the natural source is aTheobroma or Herrania species or inter- or intra-species specificcrosses thereof.
 6. The compound of claim 1 which is substantially pure.7. The compound of claim 1 which is purified to apparent homogeneity. 8.The compound of claim 1 wherein the phenolic moiety is selected from thegroup consisting of caffeic, cinnamic, coumaric, ferulic, gallic,hydroxybenzoic and sinapic acids.
 9. An antineoplastic compositioncomprising a polymeric compound of the formula A_(n), wherein A is amonomer of the formula:

wherein n is an integer from 3 to 18, such that there is at least oneterminal monomeric unit A, and a plurality of additional monomericunits; R is 3-(α)-OH, 3-(β)-OH, 3-(α)-O-sugar, or 3-(β)-O-sugar; bondingbetween adjacent monomers takes place at positions selected from thegroup consisting of 4, 6 and 8; a bond of an additional monomeric unitin position 4 has alpha or beta stereochemistry; X, Y and Z are selectedfrom the group consisting of monomeric unit A, hydrogen, and a sugar,with the provisos that as to the at least one terminal monomeric unit,bonding of the additional monomeric unit thereto is at position 4 andoptionally Y=Z=hydrogen; the sugar is optionally substituted with aphenolic moiety; pharmaceutically acceptable salts, derivatives thereof,and oxidation products thereof; and a pharmaceutically acceptablecarrier or excipient.
 10. The composition of claim 9 wherein n is 5 to12.
 11. The composition of claim 9 wherein the sugar is selected fromthe group consisting of glucose, galactose, xylose, rhamnose andarabinose.
 12. The composition of claim 9 wherein said polymericcompound is substantially pure.
 13. The composition of claim 9 whereinthe phenolic moiety is selected from the group consisting of caffeic,cinnamic, coumaric, ferulic, gallic, hydroxybenzoic and sinapic acids.14. The composition of claim 9 wherein n is
 5. 15. A method for treatinga subject in need of treatment with an antineoplastic agent comprisingadministering to the subject an antineoplastic composition as claimed inclaim
 9. 16. The method of claim 15, further comprising administering atleast one additional antineoplastic agent.
 17. A kit for a compositionof claim 9 comprising the compound and the carrier or diluent separatelypackaged, and optionally instructions for admixture or administration.18. A composition as claimed in claim 9 wherein the composition isselected from the group consisting of a tablet, capsule, Standard ofIdentity chocolate and non-Standard of Identity chocolate.
 19. Anantioxidant composition comprising a polymeric compound of the formulaA_(n), wherein A is a monomer of the formula:

wherein n is an integer from 2 to 18, such that there is at least oneterminal monomeric unit A, and one or a plurality of additionalmonomeric units; R is 3-(α)-OH, 3-(β)-OH, 3-(α)-O-sugar, or3-(β)-O-sugar; bonding between adjacent monomers takes place atpositions selected from the group consisting of 4, 6 and 8; a bond of anadditional monomeric unit in position 4 has alpha or betastereochemistry; X, Y and Z are selected from the group consisting ofmonomeric unit A, hydrogen, and a sugar, with the provisos that as tothe at least one terminal monomeric unit, bonding of the additionalmonomeric unit thereto is at position 4 and optionally Y=Z=hydrogen; thesugar is optionally substituted with a phenolic moiety; pharmaceuticallyacceptable salts, derivatives thereof, and oxidation products thereof;and a pharmaceutically acceptable carrier or excipient.
 20. Thecomposition of claim 19 wherein n is 2 to
 10. 21. The composition ofclaim 19 wherein the sugar is selected from the group consisting ofglucose, galactose, xylose, rhamnose and arabinose.
 22. The compositionof claim 19 wherein said polymeric compound is substantially pure. 23.The composition of claim 19 wherein the phenolic moiety is selected fromthe group consisting of caffeic, cinnamic, coumaric, ferulic, gallic,hydroxybenzoic and sinapic acids.
 24. The composition of claim 19wherein n is 2 to
 12. 25. A method for treating a subject in need oftreatment with an antioxidant agent comprising administering to thesubject an antioxidant composition as claimed in claim
 19. 26. Themethod of claim 25, further comprising administering at least oneadditional antioxidant agent.
 27. A composition as claimed in claim 19wherein the composition is selected from the group consisting of atablet, capsule, Standard of Identity chocolate and non-Standard ofIdentity chocolate.
 28. A kit for a composition of claim 19 comprisingthe compound and the carrier or diluent separately packaged, andoptionally instructions for admixture or administration.
 29. A methodfor preserving or protecting a desired item from oxidation comprisingcontacting the item with a composition as claimed in claim
 19. 30. Anantimicrobial composition comprising a polymeric compound of the formulaA_(n), wherein A is a monomer of the formula:

wherein n is an integer from 2 to 18, such that there is at least oneterminal monomeric unit A, and one or a plurality of additionalmonomeric units; R is 3-(α)-OH, 3-(β)-OH, 3-(α)-O-sugar, or3-(β)-O-sugar; bonding between adjacent monomers takes place atpositions selected from the group consisting of 4, 6 and 8; a bond of anadditional monomeric unit in position 4 has alpha or betastereochemistry; X, Y and Z are selected from the group consisting ofmonomeric unit A, hydrogen, and a sugar, with the provisos that as tothe at least one terminal monomeric unit, bonding of the additionalmonomeric unit thereto is at position 4 and optionally Y=Z=hydrogen; thesugar is optionally substituted with a phenolic moiety; pharmaceuticallyacceptable salts, derivatives thereof, and oxidation products thereof;and a pharmaceutically acceptable carrier or excipient.
 31. Thecomposition of claim 30 wherein n is 2 to
 10. 32. The composition ofclaim 30 wherein the sugar is selected from the group consisting ofglucose, galactose, xylose, rhamnose and arabinose.
 33. The compositionof claim 30 wherein said polymeric compound is substantially pure. 34.The composition of claim 30 wherein the phenolic moiety is selected fromthe group consisting of caffeic, cinnamic, coumaric, ferulic, gallic,hydroxybenzoic and sinapic acids.
 35. The composition of claim 30wherein n is selected from the group consisting of 2, 4, 5, 6, 8 and 10.36. A composition as claimed in claim 30 wherein the composition isselected from the group consisting of a tablet, capsule, Standard ofIdentity chocolate and non-Standard of Identity chocolate.
 37. A methodfor treating a subject in need of treatment with an antimicrobial agentcomprising administering to the subject an antimicrobial composition asclaimed in claim
 30. 38. The method of claim 37, further comprisingadministering at least one additional antimicrobial agent.
 39. A kit fora composition of claim 30 comprising the compound and the carrier ordiluent separately packaged, and optionally instructions for admixtureor administration.
 40. A cyclo-oxygenase and/or lipoxygenase modulatingcomposition comprising a polymeric compound of the formula A_(n),wherein A is a monomer of the formula:

wherein n is an integer from 2 to 18, such that there is at least oneterminal monomeric unit A, and one or a plurality of additionalmonomeric units; R is 3-(α)-OH, 3-(β)-OH, 3-(α)-O-sugar, or3-(β)-O-sugar; bonding between adjacent monomers takes place atpositions selected from the group consisting of 4, 6 and 8; a bond of anadditional monomeric unit in position 4 has alpha or betastereochemistry; X, Y and Z are selected from the group consisting ofmonomeric unit A, hydrogen, and a sugar, with the provisos that as tothe at least one terminal monomeric unit, bonding of the additionalmonomeric unit thereto is at position 4 and optionally Y=Z=hydrogen; thesugar is optionally substituted with a phenolic moiety; pharmaceuticallyacceptable salts, derivatives thereof, and oxidation products thereof;and a pharmaceutically acceptable carrier or excipient.
 41. Thecomposition of claim 40 wherein n is 2 to
 10. 42. The composition ofclaim 40 wherein the sugar is selected from the group consisting ofglucose, galactose, xylose, rhamnose and arabinose.
 43. The compositionof claim 40 wherein said polymeric compound is substantially pure. 44.The composition of claim 40 wherein the phenolic moiety is selected fromthe group consisting of caffeic, cinnamic, coumaric, ferulic, gallic,hydroxybenzoic and sinapic acids.
 45. The composition of claim 40wherein n is 2 to
 5. 46. A composition as claimed in claim 40 whereinthe composition is selected from the group consisting of a tablet,capsule, Standard of Identity chocolate and non-Standard of Identitychocolate.
 47. A method for treating a subject in need of treatment witha cyclo-oxygenase and/or lipoxygenase modulating agent comprisingadministering to the subject a composition as claimed in claim
 40. 48.The method of claim 47, further comprising administering at least oneadditional cyclo-oxygenase and/or lipoxygenase modulating agent.
 49. Akit for a composition of claim 40 comprising the compound and thecarrier or diluent separately packaged, and optionally instructions foradmixture or administration.
 50. An NO or NO-synthase modulatingcomposition comprising a polymeric compound of the formula A_(n),wherein A is a monomer of the formula:

wherein n is an integer from 2 to 18, such that there is at least oneterminal monomeric unit A, and one or a plurality of additionalmonomeric units; R is 3-(α)-OH, 3-(β)-OH, 3-(α)-O-sugar, or3-(β)-O-sugar; bonding between adjacent monomers takes place atpositions selected from the group consisting of 4, 6 and 8; a bond of anadditional monomeric unit in position 4 has alpha or betastereochemistry; X, Y and Z are selected from the group consisting ofmonomeric unit A, hydrogen, and a sugar, with the provisos that as tothe at least one terminal monomeric unit, bonding of the additionalmonomeric unit thereto is at position 4 and optionally Y=Z=hydrogen; thesugar is optionally substituted with a phenolic moiety via an esterbond; pharmaceutically acceptable salts, derivatives thereof, andoxidation products thereof; and a pharmaceutically acceptable carrier orexcipient.
 51. The composition of claim 50 wherein n is 2 to
 10. 52. Thecomposition of claim 50 wherein the sugar is selected from the groupconsisting of glucose, galactose, xylose, rhamnose and arabinose. 53.The composition of claim 50 wherein said polymeric compound issubstantially pure.
 54. The composition of claim 50 wherein the phenolicmoiety is selected from the group consisting of caffeic, cinnamic,coumaric, ferulic, gallic, hydroxybenzoic and sinapic acids.
 55. Acomposition as claimed in claim 50 wherein the composition is selectedfrom the group consisting of a tablet, capsule, Standard of Identitychocolate and non-Standard of Identity chocolate.
 56. A method fortreating a subject in need of treatment with a NO-modulating agentcomprising administering to the subject an NO or NO-synthase modulatingcomposition as claimed in claim
 50. 57. The method of claim 56, furthercomprising administering at least one additional NO or NO-synthasemodulating agent.
 58. A kit for a composition of claim 50 comprising thecompound and the carrier or diluent separately packaged, and optionallyinstructions for admixture or administration.
 59. A method of treating amammal in need of treatment with an NO or NO synthase agent comprisingadministering to the mammal a composition comprising a polymericcompound of the formula A_(n), wherein A is a monomer of the formula:

wherein n is an integer from 2 to 18, such that there is at least oneterminal monomeric unit A, and one or a plurality of additionalmonomeric units; R is 3-(α)-OH, 3-(β)-OH, 3-(α)-O-sugar, or3-(β)-O-sugar; bonding between adjacent monomers takes place atpositions selected from the group consisting of 4, 6 and 8; a bond of anadditional monomeric unit in position 4 has alpha or betastereochemistry; X, Y and Z are selected from the group consisting ofmonomeric unit A, hydrogen, and a sugar, with the provisos that as tothe at least one terminal monomeric unit, bonding of the additionalmonomeric unit thereto is at position 4 and optionally Y=Z=hydrogen; thesugar is optionally substituted with a phenolic moiety; pharmaceuticallyacceptable salts, derivatives thereof, and oxidation products thereof;and a pharmaceutically acceptable carrier or excipient.
 60. A method ofreducing NO affected hypercholesterolemia in a mammal comprisingadministering to said mammal a composition comprising a polymericcompound of the formula A_(n), wherein A is a monomer of the formula:

wherein n is an integer from 2 to 18, such that there is at least oneterminal monomeric unit A, and one or a plurality of additionalmonomeric units; R is 3-(α)-OH, 3-(β)-OH, 3-(α)-O-sugar, or3-(β)-O-sugar; bonding between adjacent monomers takes place atpositions selected from the group consisting of 4, 6 and 8; a bond of anadditional monomeric unit in position 4 has alpha or betastereochemistry; X, Y and Z are selected from the group consisting ofmonomeric unit A, hydrogen, and a sugar, with the provisos that as tothe at least one terminal monomeric unit, bonding of the additionalmonomeric unit thereto is at position 4 and optionally Y=Z=hydrogen; thesugar is optionally substituted with a phenolic moiety; pharmaceuticallyacceptable salts, derivatives thereof, and oxidation products thereof;and a pharmaceutically acceptable carrier or excipient.
 61. The methodof claim 60 wherein n is 2 to
 10. 62. A method for the induction of iNOSin mammalian monocyte and/or macrophages comprising contacting saidmonocyte and/or macrophages with a composition comprising a polymericcompound of the formula A_(n), wherein A is a monomer of the formula:

wherein n is an integer from 2 to 18, such that there is at least oneterminal monomeric unit A, and one or a plurality of additionalmonomeric units; R is 3-(α)-OH, 3-(β)-OH, 3-(α)-O-sugar, or3-(β)-O-sugar; bonding between adjacent monomers takes place atpositions selected from the group consisting of 4, 6 and 8; a bond of anadditional monomeric unit in position 4 has alpha or betastereochemistry; X, Y and Z are selected from the group consisting ofmonomeric unit A, hydrogen, and a sugar, with the provisos that as tothe at least one terminal monomeric unit, bonding of the additionalmonomeric unit thereto is at position 4 and optionally Y=Z=hydrogen; thesugar is optionally substituted with a phenolic moiety; pharmaceuticallyacceptable salts, derivatives thereof, and oxidation products thereof;and a pharmaceutically acceptable carrier or excipient.
 63. A method ofstimulating mammalian monocyte and/or macrophage NO productioncomprising administering to said mammal a composition comprising apolymeric compound of the formula A_(n), wherein A is a monomer of theformula:

wherein n is an integer from 2 to 18, such that there is at least oneterminal monomeric unit A, and one or a plurality of additionalmonomeric units; R is 3-(α)-OH, 3-(β)-OH, 3-(α)-O-sugar, or3-(β)-O-sugar; bonding between adjacent monomers takes place atpositions selected from the group consisting of 4, 6 and 8; a bond of anadditional monomeric unit in position 4 has alpha or betastereochemistry; X, Y and Z are selected from the group consisting ofmonomeric unit A, hydrogen, and a sugar, with the provisos that as tothe at least one terminal monomeric unit, bonding of the additionalmonomeric unit thereto is at position 4 and optionally Y=Z=hydrogen; thesugar is optionally substituted with a phenolic moiety; pharmaceuticallyacceptable salts, derivatives thereof, and oxidation products thereof;and a pharmaceutically acceptable carrier or excipient.
 64. The methodof claim 63 wherein n is 2 to
 10. 65. An in vivo glucose-modulatingcomposition comprising a polymeric compound of the formula A_(n),wherein A is a monomer of the formula:

wherein n is an integer from 2 to 18, such that there is at least oneterminal monomeric unit A, and one or a plurality of additionalmonomeric units; R is 3-(α)-OH, 3-(β)-OH, 3-(α)-O-sugar, or3-(β)-O-sugar; bonding between adjacent monomers takes place atpositions selected from the group consisting of 4, 6 and 8; a bond of anadditional monomeric unit in position 4 has alpha or betastereochemistry; X, Y and Z are selected from the group consisting ofmonomeric unit A, hydrogen, and a sugar, with the provisos that as tothe at least one terminal monomeric unit, bonding of the additionalmonomeric unit thereto is at position 4 and optionally Y=Z=hydrogen; thesugar is optionally substituted with a phenolic moiety; pharmaceuticallyacceptable salts, derivatives thereof, and oxidation products thereof;and a pharmaceutically acceptable carrier or excipient.
 66. Thecomposition of claim 65 wherein n is 2 to
 10. 67. The composition ofclaim 65 wherein the sugar is selected from the group consisting ofglucose, galactose, xylose, rhamnose and arabinose.
 68. The compositionof claim 65 wherein said polymeric compound is substantially pure. 69.The composition of claim 65 wherein the phenolic moiety is selected fromthe group consisting of caffeic, cinnamic, coumaric, ferulic, gallic,hydroxybenzoic and sinapic acids.
 70. A composition as claimed in claim65 wherein the composition is selected from the group consisting of atablet, capsule, Standard of Identity chocolate and non-Standard ofIdentity chocolate.
 71. A method for treating a subject in need oftreatment with a glucose-modulating agent comprising administering tothe subject a glucose-modulating agent as claimed in claim
 65. 72. Themethod of claim 71, further comprising administering at least oneadditional glucose-modulating agent.
 73. A kit for a composition ofclaim 65 comprising the compound and the carrier or diluent separatelypackaged, and optionally instructions for admixture or administration.74. A topoisomerase II-inhibiting composition comprising a polymericcompound of the formula A_(n), wherein A is a monomer of the formula:

wherein n is an integer from 2 to 18, such that there is at least oneterminal monomeric unit A, and one or a plurality of additionalmonomeric units; R is 3-(α)-OH, 3-(β)-OH, 3-(α)-O-sugar, or3-(β)-O-sugar; bonding between adjacent monomers takes place atpositions selected from the group consisting of 4, 6 and 8; a bond of anadditional monomeric unit in position 4 has alpha or betastereochemistry; X, Y and Z are selected from the group consisting ofmonomeric unit A, hydrogen, and a sugar, with the provisos that as tothe at least one terminal monomeric unit, bonding of the additionalmonomeric unit thereto is at position 4 and optionally Y=Z=hydrogen; thesugar is optionally substituted with a phenolic moiety; pharmaceuticallyacceptable salts, derivatives thereof, and oxidation products thereof;and a pharmaceutically acceptable carrier or excipient.
 75. Thecomposition of claim 74 wherein the sugar is selected from the groupconsisting of glucose, galactose, xylose, rhamnose and arabinose. 76.The composition of claim 74 wherein said polymeric compound issubstantially pure.
 77. The composition of claim 74 wherein the phenolicmoiety is selected from the group consisting of caffeic, cinnamin,coumaric, ferulic, gallic, hydroxybenzoic and sinapic acids.
 78. Acomposition as claimed in claim 74 wherein the composition is selectedfrom the group consisting of a tablet, capsule, Standard of Identitychocolate and non-Standard of Identity chocolate.
 79. A method forinhibiting topoisomerase II which comprises contacting topoisomerase IIwith a composition as claimed in claim
 74. 80. A kit for a compositionof claim 74 comprising the compound and the carrier or diluentseparately packaged, and optionally instructions for admixture oradministration.
 81. A non-steroidal antiinflammatory compositioncomprising a polymeric compound of the formula A_(n), wherein A is amonomer of the formula:

wherein n is an integer from 2 to 18, such that there is at least oneterminal monomeric unit A, and one or a plurality of additionalmonomeric units; R is 3-(α)-OH, 3-(β)-OH, 3-(α)-O-sugar, or3-(β)-O-sugar; bonding between adjacent monomers takes place atpositions selected from the group consisting of 4, 6 and 8; a bond ofthe additional monomeric unit in position 4 has alpha or betastereochemistry; X, Y and Z are selected from the group consisting ofmonomeric unit A, hydrogen, and a sugar, with the provisos that as tothe at least one terminal monomeric unit, bonding of an additionalmonomeric unit thereto is at position 4 and optionally Y=Z=hydrogen; thesugar is optionally substituted with a phenolic moiety, andpharmaceutically acceptable salts, derivatives thereof, and oxidationproducts thereof; and a pharmaceutically acceptable carrier orexcipient.
 82. The composition of claim 81 wherein the sugar is selectedfrom the group consisting of glucose, galactose, xylose, rhamnose andarabinose.
 83. The composition of claim 81 wherein said polymericcompound is substantially pure.
 84. The composition of claim 81 whereinthe phenolic moiety is selected from the group consisting of caffeic,cinnamic, coumaric, ferulic, gallic, hydroxybenzoic and sinapic acids.85. A composition as claimed in claim 81 wherein the composition isselected from the group consisting of a tablet, capsule, Standard ofIdentity chocolate and non-Standard of Identity chocolate.
 86. A methodfor treating a subject in need of treatment with a non-steroidalantiinflammatory agent comprising administering to the subject acomposition as claimed in claim
 81. 87. The method of claim 86, furthercomprising administering at least one additional non-steroidalantiinflammatory agent.
 88. A kit for a composition of claim 81comprising the compound and the carrier or diluent separately packaged,and optionally instructions for admixture or administration.
 89. Ananti-gingivitis or anti-periodontitis composition comprising a polymericcompound of the formula A_(n), wherein A is a monomer of the formula:

wherein n is an integer from 2 to 18, such that there is at least oneterminal monomeric unit A, and one or a plurality of additionalmonomeric units; R is 3-(α)-OH, 3-(β)-OH, 3-(α)-O-sugar, or3-(β)-O-sugar; bonding between adjacent monomers takes place atpositions selected from the group consisting of 4, 6 and 8; a bond of anadditional monomeric unit in position 4 has alpha or betastereochemistry; X, Y and Z are selected from the group consisting ofmonomeric unit A, hydrogen, and a sugar, with the provisos that as tothe at least one terminal monomeric unit, bonding of the additionalmonomeric unit thereto is at position 4 and optionally Y=Z=hydrogen; thesugar is optionally substituted with a phenolic moiety; pharmaceuticallyacceptable salts, derivatives thereof, and oxidation products thereof;and a pharmaceutically acceptable carrier or excipient.
 90. Thecomposition of claim 89 wherein the sugar is selected from the groupconsisting of glucose, galactose, xylose, rhamnose and arabinose. 91.The composition of claim 89 wherein said polymeric compound issubstantially pure.
 92. The composition of claim 89 wherein the phenolicmoiety is selected from the group consisting of caffeic, cinnamic,coumaric, ferulic, gallic, hydroxybenzoic and sinapic acids.
 93. Acomposition as claimed in claim 89 wherein the composition is selectedfrom the group consisting of a tablet, capsule, Standard of Identitychocolate and non-Standard of Identity chocolate.
 94. A kit for acomposition of claim 89 comprising the compound and the carrier ordiluent separately packaged, and optionally instructions for admixtureor administration.
 95. A method of reducing periodontal diseaseprogression in a mammal comprising administering to said mammal with acomposition comprising a polymeric compound of the formula A_(n),wherein A is a monomer of the formula:

wherein n is an integer from 2 to 18, such that there is at least oneterminal monomeric unit A, and one or a plurality of additionalmonomeric units; R is 3-(α)-OH, 3-(β)-OH, 3-(α)-O-sugar, or3-(β)-O-sugar; bonding between adjacent monomers takes place atpositions selected from the group consisting of 4, 6 and 8; a bond of anadditional monomeric unit in position 4 has alpha or betastereochemistry; X, Y and Z are selected from the group consisting ofmonomeric unit A, hydrogen, and a sugar, with the provisos that as tothe at least one terminal monomeric unit, bonding of the additionalmonomeric unit thereto is at position 4 and optionally Y=Z=hydrogen; thesugar is optionally substituted with a phenolic moiety; pharmaceuticallyacceptable salts, derivatives thereof, and oxidation products thereof;and a pharmaceutically acceptable carrier or excipient.
 96. The methodof claim 95 wherein n is 2 to
 10. 97. A method of treatment ofperiodontal disease comprising administering to a mammal in need of suchtreatment a composition comprising a polymeric compound of the formulaA_(n), wherein A is a monomer of the formula:

wherein n is an integer from 2 to 18, such that there is at least oneterminal monomeric unit A, and one or a plurality of additionalmonomeric units; R is 3-(α)-OH, 3-(β)-OH, 3-(α)-O-sugar, or3-(β)-O-sugar; bonding between adjacent monomers takes place atpositions selected from the group consisting of 4, 6 and 8; a bond of anadditional monomeric unit in position 4 has alpha or betastereochemistry; X, Y and Z are selected from the group consisting ofmonomeric unit A, hydrogen, and a sugar, with the provisos that as tothe at least one terminal monomeric unit, bonding of the additionalmonomeric unit thereto is at position 4 and optionally Y=Z=hydrogen; thesugar is optionally substituted with a phenolic moiety; pharmaceuticallyacceptable salts, derivatives thereof, and oxidation products thereof;and a pharmaceutically acceptable carrier or excipient.
 98. A plateletaggregation modulating composition comprising a polymeric compound ofthe formula A_(n), wherein A is a monomer of the formula:

wherein n is an integer from 2 to 18, such that there is at least oneterminal monomeric unit A, and one or a plurality of additionalmonomeric units; R is 3-(α)-OH, 3-(β)-OH, 3-(α)-O-sugar, or3-(β)-O-sugar; bonding between adjacent monomers takes place atpositions selected from the group consisting of 4, 6 and 8; a bond of anadditional monomeric unit in position 4 has alpha or betastereochemistry; X, Y and Z are selected from the group consisting ofmonomeric unit A, hydrogen, and a sugar, with the provisos that as tothe at least one terminal monomeric unit, bonding of the additionalmonomeric unit thereto is at position 4 and optionally Y=Z=hydrogen; thesugar is optionally substituted with a phenolic moiety; pharmaceuticallyacceptable salts, derivatives thereof, and oxidation products thereof;and a pharmaceutically acceptable carrier or excipient.
 99. Thecomposition of claim 98 wherein the sugar is selected from the groupconsisting of glucose, galactose, xylose, rhamnose and arabinose. 100.The composition of claim 98 wherein said polymeric compound issubstantially pure.
 101. The composition of claim 98 wherein thephenolic moiety is selected from the group consisting of caffeic,cinnamic, coumaric, ferulic, gallic, hydroxybenzoic and sinapic acids.102. A composition as claimed in claim 98 wherein the composition isselected from the group consisting of a tablet, capsule, Standard ofIdentity chocolate and non-Standard of Identity chocolate.
 103. A kitfor a composition of claim 98 comprising the compound and the carrier ordiluent separately packaged, and optionally instructions for admixtureor administration.
 104. A method of modulating platelet aggregationadministering to a mammal in need of such treatment with a compound ofclaim
 98. 105. An apoptosis modulating composition comprising apolymeric compound of the formula A_(n), wherein A is a monomer of theformula:

wherein n is an integer from 2 to 18, such that there is at least oneterminal monomeric unit A, and one or a plurality of additionalmonomeric units; R is 3-(α)-OH, 3-(β)-OH, 3-(α)-O-sugar, or3-(β)-O-sugar; bonding between adjacent monomers takes place atpositions selected from the group consisting of 4, 6 and 8; a bond of anadditional monomeric unit in position 4 has alpha or betastereochemistry; X, Y and Z are selected from the group consisting ofmonomeric unit A, hydrogen, and a sugar, with the provisos that as tothe at least one terminal monomeric unit, bonding of the additionalmonomeric unit thereto is at position 4 and optionally Y=Z=hydrogen; thesugar is optionally substituted with a phenolic moiety; pharmaceuticallyacceptable salts, derivatives thereof, and oxidation products thereof;and a pharmaceutically acceptable carrier or excipient.
 106. Thecomposition of claim 105 wherein the sugar is selected from the groupconsisting of glucose, galactose, xylose, rhamnose and arabinose. 107.The composition of claim 105 wherein said polymeric compound issubstantially pure.
 108. The composition of claim 105 wherein thephenolic moiety is selected from the group consisting of caffeic,cinnamic, coumaric, ferulic, gallic, hydroxybenzoic and sinapic acids.109. A composition as claimed in claim 105 wherein the composition isselected from the group consisting of a tablet, capsule, Standard ofIdentity chocolate and non-Standard of Identity chocolate.
 110. A kitfor a composition of claim 105 comprising the compound and the carrieror diluent separately packaged, and optionally instructions foradmixture or administration.
 111. A method of modulating apoptosiscomprising contacting COX-2 expressing cancer cells with a compositioncomprising a polymeric compound of the formula A_(n), wherein A is amonomer of the formula:

wherein n is an integer from 2 to 18, such that there is at least oneterminal monomeric unit A, and one or a plurality of additionalmonomeric units; R is 3-(α)-OH, 3-(β)-OH, 3-(α)-O-sugar, or3-(β)-O-sugar; bonding between adjacent monomers takes place atpositions selected from the group consisting of 4, 6 and 8; a bond of anadditional monomeric unit in position 4 has alpha or betastereochemistry; X, Y and Z are selected from the group consisting ofmonomeric unit A, hydrogen, and a sugar, with the provisos that as tothe at least one terminal monomeric unit, bonding of the additionalmonomeric unit thereto is at position 4 and optionally Y=Z=hydrogen; thesugar is optionally substituted with a phenolic moiety; pharmaceuticallyacceptable salts, derivatives thereof, and oxidation products thereof;and a pharmaceutically acceptable carrier or excipient.
 112. The methodof claim 111 wherein n is
 5. 113. A method of inhibiting oxidativedamage to DNA comprising contacting DNA with a composition comprising apolymeric compound of the formula A_(n), wherein A is a monomer of theformula:

wherein n is an integer from 2 to 18, such that there is at least oneterminal monomeric unit A, and one or a plurality of additionalmonomeric units; R is 3-(α)-OH, 3-(β)-OH, 3-(α)-O-sugar, or3-(β)-O-sugar; bonding between adjacent monomers takes place atpositions selected from the group consisting of 4, 6 and 8; a bond of anadditional monomeric unit in position 4 has alpha or betastereochemistry; X, Y and Z are selected from the group consisting ofmonomeric unit A, hydrogen, and a sugar, with the provisos that as tothe at least one terminal monomeric unit, bonding of the additionalmonomeric unit thereto is at position 4 and optionally Y=Z=hydrogen; thesugar is optionally substituted with a phenolic moiety; pharmaceuticallyacceptable salts, derivatives thereof, and oxidation products thereof;and a pharmaceutically acceptable carrier or excipient.
 114. Thecomposition of claim 113 wherein the sugar is selected from the groupconsisting of glucose, galactose, xylose, rhamnose and arabinose. 115.The composition of claim 113 wherein said polymeric compound issubstantially pure.
 116. The composition of claim 113 wherein thephenolic moiety is selected from the group consisting of caffeic,cinnamic, coumaric, ferulic, gallic, hydroxybenzoic and sinapic acids.117. A composition as claimed in claim 113 wherein the composition isselected from the group consisting of a tablet, capsule, Standard ofIdentity chocolate and non-Standard of Identity chocolate.
 118. A kitfor a composition of claim 113 comprising the compound and the carrieror diluent separately packaged, and optionally instructions foradmixture or administration.
 119. A composition for treating, preventingor reducing atherosclerosis or restenosis comprising a polymericcompound of the formula A_(n), wherein A is a monomer of the formula:

wherein n is an integer from 2 to 18, such that there is at least oneterminal monomeric unit A, and one or a plurality of additionalmonomeric units; R is 3-(α)-OH, 3-(β)-OH, 3-(α)-O-sugar, or3-(β)-O-sugar; bonding between adjacent monomers takes place atpositions selected from the group consisting of 4, 6 and 8; a bond of anadditional monomeric unit in position 4 has alpha or betastereochemistry; X, Y and Z are selected from the group consisting ofmonomeric unit A, hydrogen, and a sugar, with the provisos that as tothe at least one terminal monomeric unit, bonding of the additionalmonomeric unit thereto is at position 4 and optionally Y=Z=hydrogen; thesugar is optionally substituted with a phenolic moiety; pharmaceuticallyacceptable salts, derivatives thereof, and oxidation products thereof;and a pharmaceutically acceptable carrier or excipient.
 120. Thecomposition of claim 119 wherein the sugar is selected from the groupconsisting of glucose, galactose, xylose, rhamnose and arabinose. 121.The composition of claim 119 wherein said polymeric compound issubstantially pure.
 122. The composition of claim 119 wherein thephenolic moiety is selected from the group consisting of caffeic,cinnamic, coumaric, ferulic, gallic, hydroxybenzoic and sinapic acids.123. A composition as claimed in claim 119 wherein the composition isselected from the group consisting of a tablet, capsule, Standard ofIdentity chocolate and non-Standard of Identity chocolate.
 124. A methodof treating, preventing or reducing atherosclerosis or restenosis in amammal comprising administering to said mammal a composition comprisinga polymeric compound of the formula A_(n), wherein A is a monomer of theformula:

wherein n is an integer from 2 to 18, such that there is at least oneterminal monomeric unit A, and one or a plurality of additionalmonomeric units; R is 3-(α)-OH, 3-(β)-OH, 3-(α)-O-sugar, or3-(β)-O-sugar; bonding between adjacent monomers takes place atpositions selected from the group consisting of 4, 6 and 8; a bond of anadditional monomeric unit in position 4 has alpha or betastereochemistry; X, Y and Z are selected from the group consisting ofmonomeric unit A, hydrogen, and a sugar, with the provisos that as tothe at least one terminal monomeric unit, bonding of the additionalmonomeric unit thereto is at position 4 and optionally Y=Z=hydrogen; thesugar is optionally substituted with a phenolic moiety; pharmaceuticallyacceptable salts, derivatives thereof, and oxidation products thereof;and a pharmaceutically acceptable carrier or excipient.
 125. The methodof claim 124 wherein n is 2 to
 10. 126. A method of inhibiting theoxidation of LDL in a mammal comprising administering to said mammal acomposition comprising a polymeric compound of the formula A_(n),wherein A is a monomer of the formula:

wherein n is an integer from 2 to 18, such that there is at least oneterminal monomeric unit A, and one or a plurality of additionalmonomeric units; R is 3-(α)-OH, 3-(β)-OH, 3-(α)-O-sugar, or3-(β)-O-sugar; bonding between adjacent monomers takes place atpositions selected from the group consisting of 4, 6 and 8; a bond of anadditional monomeric unit in position 4 has alpha or betastereochemistry; X, Y and Z are selected from the group consisting ofmonomeric unit A, hydrogen, and a sugar, with the provisos that as tothe at least one terminal monomeric unit, bonding of the additionalmonomeric unit thereto is at position 4 and optionally Y=Z=hydrogen; thesugar is optionally substituted with a phenolic moiety; pharmaceuticallyacceptable salts, derivatives thereof, and oxidation products thereof;and a pharmaceutically acceptable carrier or excipient.
 127. The methodof claim 126 wherein n is 2 to
 12. 128. A method of modulatingthrombosis in a mammal comprising administering to said mammal acompound of claim 1 and a suitable carrier or diluent.
 129. The methodof claim 128 wherein n is 2 to
 12. 130. A method of modulatingcyclooxygenase-1 (COX-1) for the treatment for inflammatory boweldisease comprising administering to a mammal in need of such treatment acomposition comprising a polymeric compound of the formula A_(n),wherein A is a monomer of the formula:

wherein n is an integer from 2 to 18, such that there is at least oneterminal monomeric unit A, and one or a plurality of additionalmonomeric units; R is 3-(α)-OH, 3-(β)-OH, 3-(α)-O-sugar, or3-(β)-O-sugar; bonding between adjacent monomers takes place atpositions selected from the group consisting of 4, 6 and 8; a bond of anadditional monomeric unit in position 4 has alpha or betastereochemistry; X, Y and Z are selected from the group consisting ofmonomeric unit A, hydrogen, and a sugar, with the provisos that as tothe at least one terminal monomeric unit, bonding of the additionalmonomeric unit thereto is at position 4 and optionally Y=Z=hydrogen; thesugar is optionally substituted with a phenolic moiety; pharmaceuticallyacceptable salts, derivatives thereof, and oxidation products thereof;and a pharmaceutically acceptable carrier or excipient.
 131. A method ofinhibiting bacterial growth in a mammal comprising administering to saidmammal a composition comprising a polymeric compound of the formulaA_(n), wherein A is a monomer of the formula:

wherein n is an integer from 2 to 18, such that there is at least oneterminal monomeric unit A, and one or a plurality of additionalmonomeric units; R is 3-(α)-OH, 3-(β)-OH, 3-(α)-O-sugar, or3-(β)-O-sugar; bonding between adjacent monomers takes place atpositions selected from the group consisting of 4, 6 and 8; a bond of anadditional monomeric unit in position 4 has alpha or betastereochemistry; X, Y and Z are selected from the group consisting ofmonomeric unit A, hydrogen, and a sugar, with the provisos that as tothe at least one terminal monomeric unit, bonding of the additionalmonomeric unit thereto is at position 4 and optionally Y=Z=hydrogen; thesugar is optionally substituted with a phenolic moiety; pharmaceuticallyacceptable salts, derivatives thereof, and oxidation products thereof;and a pharmaceutically acceptable carrier or excipient.
 132. The methodof claim 131 wherein n is 2, 4, 5, 6, 8 and
 10. 133. A method ofreducing hypertension in a mammal in need of such treatment comprisingadministering to said mammal a composition comprising a polymericcompound of the formula A_(n), wherein A is a monomer of the formula:

wherein n is an integer from 2 to 18, such that there is at least oneterminal monomeric unit A, and one or a plurality of additionalmonomeric units; R is 3-(α)-OH, 3-(β)-OH, 3-(α)-O-sugar, or3-(β)-O-sugar; bonding between adjacent monomers takes place atpositions selected from the group consisting of 4, 6 and 8; a bond of anadditional monomeric unit in position 4 has alpha or betastereochemistry; X, Y and Z are selected from the group consisting ofmonomeric unit A, hydrogen, and a sugar, with the provisos that as tothe at least one terminal monomeric unit, bonding of the additionalmonomeric unit thereto is at position 4 and optionally Y=Z=hydrogen; thesugar is optionally substituted with a phenolic moiety; pharmaceuticallyacceptable salts, derivatives thereof, and oxidation products thereof;and a pharmaceutically acceptable carrier or excipient.
 134. The methodof claim 133 wherein n is 2 to
 10. 135. A method for treating cancer ina mammal in need of such treatment, comprising administering to themammal in an amount sufficient to effect said treatment of at least onecompound in claim
 1. 136. The method of claim 135 wherein said cancer isprostate cancer.
 137. The method of claim 135 wherein said cancer isrenal cancer.
 138. The method of claim 135 wherein said cancer is coloncancer.
 139. The method of claim 135 wherein said cancer is breastcancer.
 140. The method of claim 135 wherein the cancer is felineleukemia.
 141. The method of claim 135 wherein the cancer is cervicalcancer.
 142. The method of claim 135 wherein the cancer is T-cellleukemia.
 143. A method of arresting cancer cell growth in a mammalcomprising administering to said mammal a compound of claim 1 and asuitable carrier or diluent.
 144. The method of claim 143 wherein n is5.
 145. A carrier or vehicle for a pharmaceutical comprising a cocoaextract.
 146. A substantially pure polyphenol from Theobroma or Herraniaspecies or inter- or intra-species specific crosses thereof comprisingpolyphenols comprising oligomers 3 through
 18. 147. A compound of claim1 having a structure giving an NMR spectra as set forth in FIG. 48A-D.148. A compound of claim 1 having a structure giving an NMR spectra asset forth in FIG. 49A-D.
 149. A method for enhancing the concentrationlevels and distribution of cocoa procyanidins in cocoa beans bymanipulating fermentation conditions.
 150. The compound as claimed inclaim 1 wherein the compound is a trimer of formula [EC-(4β→8)]₂-EC.151. The compound as claimed in claim 1 wherein the compound is atetramer of formula [EC-(4β→8)]₃-EC.
 152. The compound as claimed inclaim 1 wherein the compound is a pentamer of formula [EC-(4β→8)]₄-EC,wherein a 3-position of the terminal monomeric unit of the pentamer isoptionally derivatized with a gallate and/or β-D-glucose.
 153. Thecompound as claimed in claim 1 wherein the compound is a hexamer offormula [EC-(4β→8)]₅-EC.
 154. The compound as claimed in claim 1 whereinthe compound is a heptamer of formula [EC-(4β→8)]₆-EC.
 155. The compoundas claimed in claim 1 wherein the compound is an octamer of formula[EC-(4β→8)]₇-EC.
 156. The compound as claimed in claim 1 wherein thecompound is a nonamer of formula [EC-(4β→8)]₈-EC.
 157. The compound asclaimed in claim 1 wherein the compound is a decamer of formula[EC-(4β→8)]₉-EC.
 158. The compound as claimed in claim 1 wherein thecompound is an undecamer of formula [EC-(4β→8)]₁₀-EC.
 159. The compoundas claimed in claim 1 wherein the compound is a dodecamer of formula[EC-(4β→8)]₁₁-EC.
 160. The composition as claimed in claim 9 wherein thecompound is selected from the group consisting of a pentamer of formula[EC-(4β→8)]₄-EC, a hexamer of formula [EC-(4β→8)]₅-EC, a heptamer offormula [EC-(4β→8)]₆-EC, an octamer of formula [EC-(4β→8)]₇-EC, anonamer of formula [EC-(4β→8)]₈-EC, a decamer of formula[EC-(4β→8)]₉-EC, an undecamer of formula [EC-(4β→8)]₁₀-EC, and adodecamer of formula [EC-(4β→8)]₁₁-EC.
 161. The composition as claimedin claim 19 wherein the compound is selected from the group consistingof a dimer of formula EC-(4β→8)-EC, a trimer of formula [EC-(4β→8)]₂-EC,a tetramer of formula [EC-(4β→8)]₃-EC, a pentamer of formula[EC-(4β→8)]₄-EC, a hexamer of formula [EC-(4β→8)]₅-EC, a heptamer offormula [EC-(4β→8)]₆-EC, an octamer of formula [EC-(4β→8)]₇-EC, anonamer of formula [EC-(4β→8)]₈-EC, and a decamer of formula[EC-(4β→8)]₉-EC.
 162. The composition as claimed in claim 30 wherein thecompound is selected from the group consisting of a dimer of formulaEC-(4β→8)-EC, a trimer of formula [EC-(4β→8)]₂-EC, a tetramer of formula[EC-(4β→8)]₃-EC, a pentamer of formula [EC-(4β→8)]₄-EC, a hexamer offormula [EC-(4β→8)]₅-EC, a heptamer of formula [EC-(4β→8)]₆-EC, anoctamer of formula [EC-(4β→8)]₇-EC, a nonamer of formula[EC-(4β→8)]₈-EC, and a decamer of formula [EC-(4β→8)]₉-EC.
 163. Thecomposition as claimed in claim 40 wherein the compound is selected fromthe group consisting of a dimer of formula EC-(4β→8)-EC, a trimer offormula [EC-(4β→8)]₂-EC, a tetramer of formula [EC-(4β→8)]₃-EC, apentamer of formula [EC-(4β→8)]₄-EC, a hexamer of formula[EC-(4β→8)]₅-EC, a heptamer of formula [EC-(4β→8)]₆-EC, an octamer offormula [EC-(4β→8)]₇-EC, a nonamer of formula [EC-(4538)]₈-EC, and adecamer of formula [EC-(4β→8)]₉-EC.
 164. The composition as claimed inclaim 50 wherein the compound is selected from the group consisting of adimer of formula EC-(4β→8)-EC, a trimer of formula [EC-(4β→8)]₂-EC, atetramer of formula [EC-(4β→8)]₃-EC, a pentamer of formula[EC-(4β→8)]₄-EC, a hexamer of formula [EC-(4β→8)]₅-EC, a heptamer offormula [EC-(4β→8)]₆-EC, an octamer of formula [EC-(4β→8)]₇-EC, anonamer of formula [EC-(4β→8)]₈-EC, and a decamer of formula[EC-(4β→8)]₉-EC.
 165. The composition as claimed in claim 65 wherein thecompound is selected from the group consisting of a dimer of formulaEC-(4β→8)-EC, a trimer of formula [EC-(4β→8)]₂-EC, a tetramer of formula[EC-(4β→8)]₃-EC, a pentamer of formula [EC-(4β→8)]₄-EC, a hexamer offormula [EC-(4β→8)]₅-EC, a heptamer of formula [EC-(4β→8)]₆-EC, anoctamer of formula [EC-(4β→8)]₇-EC, a nonamer of formula[EC-(4β→8)]₈-EC, and a decamer of formula [EC-(4β→8)]₉-EC.
 166. Thecomposition as claimed in claim 74 wherein the compound is selected fromthe group consisting of a dimer of formula EC-(4β→8)-EC, a trimer offormula [EC-(4β→8)]₂-EC, a tetramer of formula [EC-(4β→8)]₃-EC, apentamer of formula [EC-(4β→8)]₄-EC, a hexamer of formula[EC-(4β→8)]₅-EC, a heptamer of formula [EC-(4β→8)]₆-EC, an octamer offormula [EC-(4β→8)]₇-EC, a nonamer of formula [EC-(4β→8)]₈-EC, a decamerof formula [EC-(4β→8)]₉-EC, an undecamer of formula [EC-(4β→8)]₁₀-EC,and a dodecamer of formula [EC-(4β→8)]₁₁-EC.
 167. The composition asclaimed in claim 81 wherein the compound is selected from the groupconsisting of a dimer of formula EC-(4β→8)-EC, a trimer of formula[EC-(4β→8)]₂-EC, a tetramer of formula [EC-(4β→8)]₃-EC, a pentamer offormula [EC-(4β→8)]₄-EC, a hexamer of formula [EC-(4β→8)]₅-EC, aheptamer of formula [EC-(4β→8)]₆-EC, an octamer of formula[EC-(4β→8)]₇-EC, a nonamer of formula [EC-(4β→8)]₈-EC, a decamer offormula [EC-(4β→8)]₉-EC, an undecamer of formula [EC-(4β→8)]₁₀-EC, and adodecamer of formula [EC-(4β→8)]₁₁-EC.
 168. The composition as claimedin claim 89 wherein the compound is selected from the group consistingof a dimer of formula EC-(4β→8)-EC, a trimer of formula [EC-(4β→8)]₂-EC,a tetramer of formula [EC-(4β→8)]₃-EC, a pentamer of formula[EC-(4β→8)]₄-EC, a hexamer of formula [EC-(4β→8)]₅-EC, a heptamer offormula [EC-(4β→8)]₆-EC, an octamer of formula [EC-(4β→8)]₇-EC, anonamer of formula [EC-(4β→8)]₈-EC, and a decamer of formula[EC-(4β→8)]₉-EC.
 169. The composition as claimed in claim 98 wherein thecompound is selected from the group consisting of a dimer of formulaEC-(4β→8)-EC, a trimer of formula [EC-(4β→8)]₂-EC, a tetramer of formula[EC-(4β→8)]₃-EC, a pentamer of formula [EC-(4β→8)]₄-EC, a hexamer offormula [EC-(4β→8)]₅-EC, a heptamer of formula [EC-(4→β8)]₆-EC, anoctamer of formula [EC-(4β→8)]₇-EC, a nonamer of formula[EC-(4β→8)]₈-EC, a decamer of formula [EC-(4β→8)]₉-EC, an undecamer offormula [EC-(4β→8)]₁₀-EC, and a dodecamer of formula [EC-(4β→8)]₁₁-EC.170. The composition as claimed in claim 105 wherein the compound is apentamer of formula [EC-(4β→8)]₄-EC.
 171. The compositition as claimedin claim 119 wherein the compound is selected from the group consistingof a dimer of formula EC-(4β→8)-EC, a trimer of formula [EC-(4β→8)]₂-EC,a tetramer of formula [EC-(4β→8)]₃-EC, a pentamer of formula[EC-(4β→8)]₄-EC, a hexamer of formula [EC-(4β→8)]₅-EC, a heptamer offormula [EC-(4β→8)]₆-EC, an octamer of formula [EC-(4β→8)]₇-EC, anonamer of formula [EC-(4β→8)]₈-EC, and a decamer of formula[EC-(4β→8)]₉-EC.
 172. A method for the identification of at least onegene induced or repressed by a polymeric compound of the formula A_(n),wherein A is a monomer of the formula:

wherein n is an integer from 2 to 18, such that there is at least oneterminal monomeric unit A, and one or a plurality of additionalmonomeric units; R is 3-(α)-OH, 3-(β)-OH, 3-(α)-O-sugar, or3-(β)-O-sugar; bonding between adjacent monomers takes place atpositions selected from the group consisting of 4, 6 and 8; a bond foran additional monomeric unit in position 4 has alpha or betastereochemistry; X, Y and Z are selected from the group consisting ofmonomeric unit A, hydrogen, and a sugar, with the provisos that as tothe at least one terminal monomeric unit, bonding of the additionalmonomeric unit thereto is at position 4 and optionally Y=Z=hydrogen; thesugar is optionally substituted with a phenolic moiety, andpharmaceutically acceptable salts, derivatives thereof, oxidationproducts thereof, or combinations thereof, said method comprisingcontacting said at least one gene or a gene product thereof with thepolymeric compound using a gene expression assay.
 173. The method asclaimed in claim 172 wherein said gene expression assay is selected fromthe group consisting of Differential Display, sequencing of cDNAlibraries, Serial Analysis of Gene Expression, and expression monitoringby hybridization to high density oligonucleotide arrays.
 174. Thecompound as claimed in claim 1, wherein adjacent monomers bind atposition 4 by (4→6) or (4→8); and each of X, Y and Z is H, a sugar or anadjacent monomer, with the provisos that if X and Y are adjacentmonomers, Z is H or sugar and if X and Z are adjacent monomers, Y is Hor sugar, and that as to at least one of the two terminal monomers,bonding of the adjacent monomer is at position 4 and optionally,Y=Z=hydrogen.
 175. The composition as claimed in claim 9, wherein thecompound further comprises adjacent monomers bind at position 4 by (4→6)or (4→8); and each of X, Y and Z is H, a sugar or an adjacent monomer,with the provisos that if X and Y are adjacent monomers, Z is H or sugarand if X and Z are adjacent monomers, Y is H or sugar, and that as to atleast one of the two terminal monomers, bonding of the adjacent monomeris at position 4 and optionally, Y=Z=hydrogen.
 176. The composition asclaimed in claim 19, wherein the compound further comprises adjacentmonomers bind at position 4 by (4→6) or (4→8); and each of X, Y and Z isH, a sugar or an adjacent monomer, with the provisos that if X and Y areadjacent monomers, Z is H or sugar and if X and Z are adjacent monomers,Y is H or sugar, and that as to at least one of the two terminalmonomers, bonding of the adjacent monomer is at position 4 andoptionally, Y=Z=hydrogen.
 177. The composition as claimed in claim 30,wherein the compound further comprises adjacent monomers bind atposition 4 by (4→6) or (4→8); and each of X, Y and Z is H, a sugar or anadjacent monomer, with the provisos that if X and Y are adjacentmonomers, Z is H or sugar and if X and Z are adjacent monomers, Y is Hor sugar, and that as to at least one of the two terminal monomers,bonding of the adjacent monomer is at position 4 and optionally,Y=Z=hydrogen.
 178. The composition as claimed in claim 40, wherein thecompound further comprises adjacent monomers bind at position 4 by (4→6)or (4→8); and each of X, Y and Z is H, a sugar or an adjacent monomer,with the provisos that if X and Y are adjacent monomers, Z is H or sugarand if X and Z are adjacent monomers, Y is H or sugar, and that as to atleast one of the two terminal monomers, bonding of the adjacent monomeris at position 4 and optionally, Y=Z=hydrogen.
 179. The composition asclaimed in claim 50, wherein the compound further comprises adjacentmonomers bind at position 4 by (4→6) or (4→8); and each of X, Y and Z isH, a sugar or an adjacent monomer, with the provisos that if X and Y areadjacent monomers, Z is H or sugar and if X and Z are adjacent monomers,Y is H or sugar, and that as to at least one of the two terminalmonomers, bonding of the adjacent monomer is at position 4 andoptionally, Y=Z=hydrogen.
 180. The composition as claimed in claim 65,wherein the compound further comprises adjacent monomers bind atposition 4 by (4→6) or (4→8); and each of X, Y and Z is H, a sugar or anadjacent monomer, with the provisos that if X and Y are adjacentmonomers, Z is H or sugar and if X and Z are adjacent monomers, Y is Hor sugar, and that as to at least one of the two terminal monomers,bonding of the adjacent monomer is at position 4 and optionally,Y=Z=hydrogen.
 181. The composition as claimed in claim 74, wherein thecompound further comprises adjacent monomers bind at position 4 by (4→6)or (4→8); and each of X, Y and Z is H, a sugar or an adjacent monomer,with the provisos that if X and Y are adjacent monomers, Z is H or sugarand if X and Z are adjacent monomers, Y is H or sugar, and that as to atleast one of the two terminal monomers, bonding of the adjacent monomeris at position 4 and optionally, Y=Z=hydrogen.
 182. The composition asclaimed in claim 81, wherein the compound further comprises adjacentmonomers bind at position 4 by (4→6) or (4→8); and each of X, Y and Z isH, a sugar or an adjacent monomer, with the provisos that if X and Y areadjacent monomers, Z is H or sugar and if X and Z are adjacent monomers,Y is H or sugar, and that as to at least one of the two terminalmonomers, bonding of the adjacent monomer is at position 4 andoptionally, Y=Z=hydrogen.
 183. The composition as claimed in claim 89,wherein the compound further comprises adjacent monomers bind atposition 4 by (4→6) or (4→8); and each of X, Y and Z is H, a sugar or anadjacent monomer, with the provisos that if X and Y are adjacentmonomers, Z is H or sugar and if X and Z are adjacent monomers, Y is Hor sugar, and that as to at least one of the two terminal monomers,bonding of the adjacent monomer is at position 4 and optionally,Y=Z=hydrogen.
 184. The composition as claimed in claim 98, wherein thecompound further comprises adjacent monomers bind at position 4 by (4→6)or (4→8); and each of X, Y and Z is H, a sugar or an adjacent monomer,with the provisos that if X and Y are adjacent monomers, Z is H or sugarand if X and Z are adjacent monomers, Y is H or sugar, and that as to atleast one of the two terminal monomers, bonding of the adjacent monomeris at position 4 and optionally, Y=Z=hydrogen.
 185. The composition asclaimed in claim 105, wherein the compound further comprises adjacentmonomers bind at position 4 by (4→6) or (4→8); and each of X, Y and Z isH, a sugar or an adjacent monomer, with the provisos that if X and Y areadjacent monomers, Z is H or sugar and if X and Z are adjacent monomers,Y is H or sugar, and that as to at least one of the two terminalmonomers, bonding of the adjacent monomer is at position 4 andoptionally, Y=Z=hydrogen.
 186. The composition as claimed in claim 119,wherein the compound further comprises adjacent monomers bind atposition 4 by (4→6) or (4→8); and each of X, Y and Z is H, a sugar or anadjacent monomer, with the provisos that if X and Y are adjacentmonomers, Z is H or sugar and if X and Z are adjacent monomers, Y is Hor sugar, and that as to at least one of the two terminal monomers,bonding of the adjacent monomer is at position 4 and optionally,Y=Z=hydrogen.
 187. The compound as claimed in claim 1 wherein thecompound is a trimer selected from the group consisting of[EC-(4β→8)]₂-EC, [EC-(4β→8)]₂-C and [EC-(4β→6)]₂-EC.
 188. The compoundas claimed in claim 1 wherein the compound is a tetramer selected fromthe group consisting of [EC-(4β→8)]₃-EC, [EC-(4β→8)]₃-C and[EC-(4β→8)]₂-EC-(4β→6)-C.
 189. The compound as claimed in claim 1wherein the compound is a pentamer selected from the group consisting of[EC-(4β→8)]₄-EC, [EC-(4β→8)]₃-EC-(4β→6)-EC, [EC-(4β→8)]₃-EC-(4β→8)-C and[EC-(4β→8)]₃-EC-(4β→6)-C.
 190. The compound as claimed in claim 1wherein the compound is a hexamer selected from the group consisting of[EC-(4β→8)]₅-EC, [EC-(4β→8)]₄-EC-(4β→6)-EC, [EC-(4β→8)]₄-EC-(4β→8)-C,and [EC-(4β→8)]₄-EC-(4β→6)-C, wherein the 3-position of a hexamerterminal monomeric unit is optionally derivatized with a gallate or aβ-D-glucose.
 191. The compound as claimed in claim 1 wherein thecompound is a heptamer selected from the group consisting of[EC-(4β→8)]₆-EC, [EC-(4β→8)]₅-EC-(4β→6)-EC, [EC-(4β→8)]₅-EC-(4β→8)-C,and [EC-(4β→8)]₅-EC-(4β→6)-C, wherein the 3-position of the heptamerterminal monomeric unit is optionally derivatized with a gallate or aβ-D-glucose.
 192. The compound as claimed in claim 1 wherein thecompound is an octamer selected from the group consisting of[EC-(4β→8)]₇-EC, [EC-(4β→8)]₆-EC-(4β→6)-EC, [EC-(4β→8)]₆-EC-(4β→8)-C,and [EC-(4β→8)]₆-EC-(4β→6)-C, wherein the 3-position of an octamerterminal monomeric unit is optionally derivatized with a gallate or aβ-D-glucose.
 193. The compound as claimed in claim 1 wherein thecompound is a nonamer selected from the group consisting of[EC-(4β→8)]₈-EC, [EC-(4β→8)]₇-EC-(4β→6)-EC, [EC-(4β→8)]₇-EC-(4β→8)-C,and [EC-(4β→8)]₇-EC-(4β→6)-C, wherein the 3-position of a nonamerterminal monomeric unit is optionally derivatized with a gallate or aβ-D-glucose.
 194. The compound as claimed in claim 1 wherein thecompound is a decamer selected from the group consisting of[EC-(4β→8)]₉-EC, [EC-(4β→8)]₈-EC-(4β→6)-EC, [EC-(4β→8)]₈-EC-(4β→8)-C,and [EC-(4β→8)]₈-EC-(4β→6)-C, wherein the 3-position of a decamerterminal monomeric unit is optionally derivatized with a gallate or aβ-D-glucose.
 195. The compound as claimed in claim 1 wherein thecompound is an undecamer selected from the group consisting of[EC-(4β→8)]₁₀-EC, [EC-(4β→8)]₉-EC-(4β→6)-EC, [EC-(4β→8)]₉-EC-(4β→8)-C,and [EC-(4β→8)]₉-EC-(4β→6)-C, wherein the 3-position of an undecamerterminal monomeric unit is optionally derivatized with a gallate or aβ-D-glucose.
 196. The compound as claimed in claim 1 wherein thecompound is a dodecamer selected from the group consisting of[EC-(4β→8)]₁₁-EC, [EC-(4β→8)]₁₀-EC-(4β→6)-EC, [EC-(4β→8)]₁₀-EC-(4β→8)-C,and [EC-(4β→8)]₁₀-EC-(4β→6)-C, wherein the 3-position of a dodecamerterminal monomeric unit is optionally derivatized with a gallate or aβ-D-glucose.
 197. The composition as claimed in claim 9 wherein thecompound is selected from the group consisting of a pentamer selectedfrom the group consisting of [EC-(4β→8)]₄-EC,[EC-(4β→38)]₃-EC-(4β→6)-EC, [EC-(4β→8)]₃-EC-(4β→8)-C and[EC-(4β→8)]₃-EC-(4β→6)-C, a hexamer selected from the group consistingof [EC-(4β→8)]₅-EC, [EC-(4β→8)]₄-EC-(4β→6)-EC, [EC-(4β→8)]₄-EC-(4β→8)-C,and [EC-(4β→8)]₄-EC-(4β→6)-C, a heptamer selected from the groupconsisting of [EC-(4β→8)]₆-EC, [EC-(4β→8)]₅-EC-(4β→6)-EC,[EC-(4β→8)]₅-EC-(4β→8)-C, and [EC-(4β→8)]₅-EC-(4β→6)-C, an octamerselected from the group consisting of [EC-(4β→8)]₇-EC,[EC-(4β→8)]₆-EC-(4β→6)-EC, [EC-(4β→8)]₆-EC-(4β→8)-C, and[EC-(4β→8)]₆-EC-(4β→6)-C, a nonamer selected from the group consistingof [EC-(4β→8)]₈-EC, [EC-(4β→8)]₇-EC-(4β→6)-EC, [EC-(4β→8)]₇-EC-(4β→8)-C,and [EC-(4β→8)]₇-EC-(4β→6)-C, a decamer selected from the groupconsisting of [EC-(4β→8)]₉-EC, [EC-(4β→8)]₈-EC-(4β→6)-EC,[EC-(4β→8)]₈-EC-(4β→8)-C, and [EC-(4β→8)]₈-EC-(4β→6)-C, an undecamerselected from the group consisting of [EC-(4β→8)]₁₀-EC,[EC-(4β→8)]₉-EC-(4β→6)-EC, [EC-(4β→8)]₉-EC-(4β→8)-C, and[EC-(4β→8)]₉-EC-(4β→6)-C, and a dodecamer selected from the groupconsisting of [EC-(4β→8)]₁₁-EC, [EC-(4β→8)]₁₀-EC-(4β→6)-EC,[EC-(4β→8)]₁₀-EC-(4β→8)-C, and [EC-(4β→8)]₁₀-EC-(4β→6)-C.
 198. Thecomposition as claimed in claim 19 wherein the compound is selected fromthe group consisting of a trimer selected from the group consisting of[EC-(4β→8)]₂-EC, [EC-(4β→8)]₂-C and [EC-(4β→6)]₂-EC, a tetramer selectedfrom the group consisting of [EC-(4β→8)]₃-EC, [EC-(4β→8)]₃-C and[EC-(4β→8)]₂-EC-(4β→6)-C, a pentamer selected from the group consistingof [EC-(4β43 8)]₄-EC, [EC-(4β→8)]₃-EC-(4β→6)-EC,[EC-(4β→8)]₃-EC-(4β→8)-C and [EC-(4β→8)]₃-EC-(4β→6)-C, a hexamerselected from the group consisting of [EC-(4β→8)]₅-EC,[EC-(4β→8)]₄-EC-(4β→6)-EC, [EC-(4β→8)]₄-EC-(4β→8)-C, and[EC-(4β→8)]₄-EC-(4β→6)-C, a heptamer selected from the group consistingof [EC-(4β→8)]₆-EC, [EC-(4β→8)]₅-EC-(4β→6)-EC, [EC-(4β→8)]₅-EC-(4β→8)-C,and [EC-(4β→8)]₅-EC-(4β→6)-C, an octamer selected from the groupconsisting of [EC-(4β→8)]₇-EC, [EC-(4β→8)]₆-EC-(4β→6)-EC,[EC-(4β→8)]₆-EC-(4β→8)-C, and [EC-(4β→8)]₆-EC-(4β→6)-C, a nonamerselected from the group consisting of [EC-(4β→8)]₈-EC,[EC-(4β→8)]₇-EC-(4β→6)-EC, [EC-(4β→8)]₇-EC-(4β→8)-C, and[EC-(4β→8)]₇-EC-(4β→6)-C, and a decamer selected from the groupconsisting of [EC-(4β→8)]₉-EC, [EC-(4β→8)]₈-EC-(4β43 6)-EC,[EC-(4β→8)]₈-EC-(4β→8)-C, and [EC-(4β→8)]₈-EC-(4β→6)-C.
 199. Thecomposition as claimed in claim 30 wherein the compound is selected fromthe group consisting of a trimer selected from the group consisting of[EC-(4β43 8)]₂-EC, [EC-(4β→8)]₂-C and [EC-(4β→6)]₂-EC, a tetramerselected from the group consisting of [EC-(4β→8)]₃-EC, [EC-(4β→8)]₃-Cand [EC-(4β→8)]₂-EC-(4β→6)-C, a pentamer selected from the groupconsisting of [EC-(4β→8)]₄-EC, [EC-(4β→8)]₃-EC-(4β→6)-EC,[EC-(4β→8)]₃-EC-(4β→8)-C and [EC-(4β→8)]₃-EC-(4β→6)-C, a hexamerselected from the group consisting of [EC-(4β→8)]₅-EC,[EC-(4β→8)]₄-EC-(4β→6)-EC, [EC-(4β→8)]₄-EC-(4β→8)-C, and[EC-(4β→8)]₄-EC-(4β→6)-C, a heptamer selected from the group consistingof [EC-(4β→8)]₆-EC, [EC-(4β→8)]₅-EC-(4β→6)-EC, [EC-(4β→8)]₅-EC-(4β→8)-C,and [EC-(4β→8)]₅-EC-(4β→6)-C, an octamer selected from the groupconsisting of [EC-(4β→8)]₇-EC, [EC-(4β→8)]₆-EC-(4β→6)-EC,[EC-(4β→8)]₆-EC-(4β→8)-C, and [EC-(4β→8)]₆-EC-(4β→6)-C, a nonamerselected from the group consisting of [EC-(4β→8)]₈-EC,[EC-(4β→8)]₇-EC-(4β→6)-EC, [EC-(4β→8)]₇-EC-(4β→8)-C, and[EC-(4β→8)]₇-EC-(4β→6)-C, and a decamer selected from the groupconsisting of [EC-(4β→8)]₉-EC, [EC-(4β→8)]g-EC-(4β→6)-EC,[EC-(4β→8)]₈-EC-(4β→8)-C, and [EC-(4β→8)]₈-EC-(4β→6)-C.
 200. Thecomposition as claimed in claim 40 wherein the compound is selected fromthe group consisting of a trimer selected from the group consisting of[EC-(4β→8)]₂-EC, [EC-(4β→8)]₂-C and [EC-(4β→6)]₂-EC, a tetramer selectedfrom the group consisting of [EC-(4β→8)]₃-EC, [EC-(4β→8)]₃-C and[EC-(4β→8)]₂-EC-(4β→6)-C, a pentamer selected from the group consistingof [EC-(4β→8)]₄-EC, [EC-(4β→8)]₃-EC-(4β→6)-EC, [EC-(4β→8)]₃-EC-(4β→8)-Cand [EC-(4β→8)]₃-EC-(4β→6)-C, a hexamer selected from the groupconsisting of [EC-(4β→8)]₅-EC, [EC-(4β→8)]₄-EC-(4β→6)-EC,[EC-(4β→8)]₄-EC-(4β→8)-C, and [EC-(4β→8)]₄-EC-(4β→6)-C, a heptamerselected from the group consisting of [EC-(4β→8)]₆-EC,[EC-(4β→8)]₅-EC-(4β→6)-EC, [EC-(4β→8)]₅-EC-(4β→8)-C, and[EC-(4β→8)]₅-EC-(4β→6)-C, an octamer selected from the group consistingof [EC-(4β→8)]₇-EC, [EC-(4β→8)]₆-EC-(4β→6)-EC, [EC-(4β→8)]₆-EC-(4β→8)-C,and [EC-(4β→8)]₆-EC-(4β→6)-C, a nonamer selected from the groupconsisting of [EC-(4β→8)]₈-EC, [EC-(4β→8)]₇-EC-(4β→6)-EC,[EC-(4β→8)]₇-EC-(4β→8)-C, and [EC-(4β→8)]₇-EC-(4β→6)-C, and a decamerselected from the group consisting of [EC-(4β→8)]₉-EC,[EC-(4β→8)]₈-EC-(4β→6)-EC, [EC-(4β→8)]₈-EC-(4β→8)-C, and[EC-(4β→8)]₈-EC-(4β→6)-C.
 201. The composition as claimed in claim 50wherein the compound is selected from the group consisting of a trimerselected from the group consisting of [EC-(4β→8)]₂-EC, [EC-(4β→8)]₂-Cand [EC-(4β→6)]₂-EC, a tetramer selected from the group consisting of[EC-(4β→8)]₃-EC, [EC-(4β→8)]₃-C and [EC-(4β→8)]₂-EC-(4β→6)-C, a pentamerselected from the group consisting of [EC-(4β→8)]₄-EC,[EC-(4β→8)]₃-EC-(4β→6)-EC, [EC-(4β→8)]₃-EC-(4β→8)-C and[EC-(4β→8)]₃-EC-(4β→6)-C, a hexamer selected from the group consistingof [EC-(4β→8)]₅-EC, [EC-(4β→8)]₄-EC-(4β→6)-EC, [EC-(4β→8)]₄-EC-(4β→8)-C,and [EC-(4β→8)]₄-EC-(4β→6)-C, a heptamer selected from the groupconsisting of [EC-(4β→8)]₆-EC, [EC-(4β→8)]₅-EC-(4β→6)-EC,[EC-(4β→8)]₅-EC-(4β→8)-C, and [EC-(4β→8)]₅-EC-(4β→6)-C, an octamerselected from the group consisting of [EC-(4β→8)]₇-EC,[EC-(4β→8)]₆-EC-(4β→6)-EC, [EC-(4β→8)]₆-EC-(4β→8)-C, and[EC-(4β→8)]₆-EC-(4β→6)-C, a nonamer selected from the group consistingof [EC-(4β→8)]₈-EC, [EC-(4β→8)]₇-EC-(4β→6)-EC, [EC-(4β→8)]₇-EC-(4β→8)-C,and [EC-(4β→8)]₇-EC-(4β→6)-C, and a decamer selected from the groupconsisting of [EC-(4β→8)]₉-EC, [EC-(4β→8)]₈-EC-(4β→6)-EC,[EC-(4β→8)]₈-EC-(4β→8)-C, and [EC-(4β→8)]₈-EC-(4β→6)-C.
 202. Thecomposition as claimed in claim 65 wherein the compound is selected fromthe group consisting of a trimer selected from the group consisting of[EC-(4β→8)]₂-EC, [EC-(4β→8)]₂-C and [EC-(4β→6)]₂-EC, a tetramer selectedfrom the group consisting of [EC-(4β→8)]₃-EC, [EC-(4β→8)]₃-C and[EC-(4β→8)]₂-EC-(4β→6)-C, a pentamer selected from the group consistingof [EC-(4β→8)]₄-EC, [EC-(4β→8)]₃-EC-(4β→6)-EC, [EC-(4β→8)]₃-EC-(4β→8)-Cand [EC-(4β→8)]₃-EC-(4β→6)-C, a hexamer selected from the groupconsisting of [EC-(4β→8)]₅-EC, [EC-(4β→8)]₄-EC-(4β→6)-EC,[EC-(4β→8)]₄-EC-(4β→8)-C, and [EC-(4β→8)]₄-EC-(4β→6)-C, a heptamerselected from the group consisting of [EC-(4β→8)]₆-EC,[EC-(4β→8)]₅-EC-(4β→6)-EC, [EC-(4β→8)]₅-EC-(4β→8)-C, and[EC-(4β→8)]₅-EC-(4β→6)-C, an octamer selected from the group consistingof [EC-(4β→8)]₇-EC, [EC-(4β→8)]₆-EC-(4β→6)-EC, [EC-(4β→8)]₆-EC-(4β→8)-C,and [EC-(4β→8)]₆-EC-(4β→6)-C, a nonamer selected from the groupconsisting of [EC-(4β→8)]₈-EC, [EC-(4β→8)]₇-EC-(4β→6)-EC,[EC-(4β→8)]₇-EC-(4β→8)-C, and [EC-(4β→8)]₇-EC-(4β→6)-C, and a decamerselected from the group consisting of [EC-(4β→8)]₉-EC,[EC-(4β→8)]₈-EC-(4β→6)-EC, [EC-(4β→8)]₈-EC-(4β→8)-C, and[EC-(4β→8)]₈-EC-(4β→6)-C.
 203. The composition as claimed in claim 74wherein the compound is selected from the group consisting of a trimerselected from the group consisting of [EC-(4β→8)]₂-EC, [EC-(4β→8)]₂-Cand [EC-(4β→6)]₂-EC, a tetramer selected from the group consisting of[EC-(4β→8)]₃-EC, [EC-(4β→8)]₃-C and [EC-(4β→8)]₂-EC-(4β→6)-C, a pentamerselected from the group consisting of [EC-(4β→8)]₄-EC,[EC-(4β→8)]₃-EC-(4β→6)-EC, [EC-(4β→8)]₃-EC-(4β→8)-C and[EC-(4β→8)]₃-EC-(4β→6)-C, a hexamer selected from the group consistingof [EC-(4β→8)]₅-EC, [EC-(4β→8)]₄-EC-(4β→6)-EC, [EC-(4β→8)]₄-EC-(4β→8)-C,and [EC-(4β→8)]₄-EC-(4β→6)-C, a heptamer selected from the groupconsisting of [EC-(4β→8)]₆-EC, [EC-(4β→8)]₅-EC-(4β→6)-EC,[EC-(4β→8)]₅-EC-(4β→8)-C, and [EC-(4β→8)]₅-EC-(4β→6)-C, an octamerselected from the group consisting of [EC-(4β→8)]₇-EC,[EC-(4β→8)]₆-EC-(4β→6)-EC, [EC-(4β→8)]₆-EC-(4β→8)-C, and[EC-(4β→8)]₆-EC-(4β→6)-C, a nonamer selected from the group consistingof [EC-(4β→8)]₈-EC, [EC-(4β→8)]₇-EC-(4β→6)-EC, [EC-(4β→8)]₇-EC-(4β→8)-C,and [EC-(4β→8)]₇-EC-(4β→6)-C, a decamer selected from the groupconsisting of [EC-(4β→8)]₉-EC, [EC-(4β→8)]₈-EC-(4β→6)-EC,[EC-(4β→8)]₈-EC-(4β→8)-C, and [EC-(4β→8)]₈-EC-(4β→6)-C, an undecamerselected from the group consisting of [EC-(4β→8)]₁₀-EC, [EC-(4β438)]₉-EC-(4β→6)-EC, [EC-(4β→8)]₉-EC-(4β→8)-C, and[EC-(4β→8)]₉-EC-(4β→6)-C, and a dodecamer selected from the groupconsisting of [EC-(4β→8)]₁₁-EC, [EC-(4β→8)]₁₀-EC-(4β→6)-EC,[EC-(4β→8)]₁₀-EC-(4β→8)-C, and [EC-(4β→8)]₁₀-EC-(4β→6)-C.
 204. Thecomposition as claimed in claim 81 wherein the compound is selected fromthe group consisting of a trimer selected from the group consisting of[EC-(4β→8)]₂-EC, [EC-(4β→8)]₂-C and [EC-(4β→6)]₂-EC, a tetramer selectedfrom the group consisting of [EC-(4β→8)]₃-EC, [EC-(4β→8)]₃-C and[EC-(4β→8)]₂-EC-(4β→6)-C, a pentamer selected from the group consistingof [EC-(4β→8)]₄-EC, [EC-(4β→8)]₃-EC-(4β→6)-EC, [EC-(4β→8)]₃-EC-(4β→8)-Cand [EC-(4β→8)]₃-EC-(4β→6)-C, a hexamer selected from the groupconsisting of [EC-(4β→8)]₅-EC, [EC-(4β→8)]₄-EC-(4β43 6)-EC,[EC-(4β→8)]₄-EC-(4β→8)-C, and [EC-(4β→8)]₄-EC-(4β→6)-C, a heptamerselected from the group consisting of [EC-(4β→8)]₆-EC,[EC-(4β→8)]₅-EC-(4β→6)-EC, [EC-(4β→8)]₅-EC-(4β→8)-C, and[EC-(4β→8)]₅-EC-(4β→6)-C, an octamer selected from the group consistingof [EC-(4β→8)]₇-EC, [EC-(4β→8)]₆-EC-(4β→6)-EC, [EC-(4β→8)]₆-EC-(4β→8)-C,and [EC-(4β→8)]₆-EC-(4β→6)-C, a nonamer selected from the groupconsisting of [EC-(4β→8)]g-EC, [EC-(4β→8)]₇-EC-(413-6)-EC,[EC-(4β→8)]₇-EC-(4β→8)-C, and [EC-(4β→8)]₇-EC-(4β→6)-C, a decamerselected from the group consisting of [EC-(4β→8)]₉-EC,[EC-(4β→8)]₈-EC-(4β→6)-EC, [EC-(4β→8)]₈-EC-(4β→8)-C, and[EC-(4β→8)]₈-EC-(4β→6)-C, an undecamer selected from the groupconsisting of [EC-(4β→8)]₁₀-EC, [EC-(4β→8)]₉-EC-(4β→6)-EC,[EC-(4β→8)]₉-EC-(4β→8)-C, and [EC-(4β→8)]₉-EC-(4β→6)-C, and a dodecamerselected from the group consisting of [EC-(4β→8)]₁₁-EC,[EC-(4β→8)]₁₀-EC-(4β→6)-EC, [EC-(4β→8)]₁₀-EC-(4β→8)-C, and[EC-(4β→8)]₁₀-EC-(4β→6)-C.
 205. The composition as claimed in claim 89wherein the compound is selected from the group consisting of a trimerselected from the group consisting of [EC-(4β→8)]₂-EC, [EC-(4β→8)]₂-Cand [EC-(4β→6)]₂-EC, a tetramer selected from the group consisting of[EC-(4β→8)]₃-EC, [EC-(4β→8)]₃-C and [EC-(4β→8)]₂-EC-(4β→6)-C, a pentamerselected from the group consisting of [EC-(4β→8)]₄-EC,[EC-(4β→8)]₃-EC-(4β→6)-EC, [EC-(4β→8)]₃-EC-(4β→8)-C and[EC-(4β→8)]₃-EC-(4β→6)-C, a hexamer selected from the group consistingof [EC-(4β→8)]₅-EC, [EC-(4β→8)]₄-EC-(4β→6)-EC, [EC-(4β→8)]₄-EC-(4β→8)-C,and [EC-(4β→8)]₄-EC-(4β→6)-C, a heptamer selected from the groupconsisting of [EC-(4β→8)]₆-EC, [EC-(4β→8)]₅-EC-(4β→6)-EC,[EC-(4β→8)]₅-EC-(4β→8)-C, and [EC-(4β→8)]₅-EC-(4β→6)-C, an octamerselected from the group consisting of [EC-(4β→8)]₇-EC,[EC-(4β→8)]₆-EC-(4β→6)-EC, [EC-(4β→8)]₆-EC-(4β→8)-C, and[EC-(4β→8)]₆-EC-(4β→6)-C, a nonamer selected from the group consistingof [EC-(4β→8)]₈-EC, [EC-(4β→8)]₇-EC-(4β→6)-EC, [EC-(4β→8)]₇-EC-(4β→8)-C,and [EC-(4β→8)]₇-EC-(4β→6)-C, and a decamer selected from the groupconsisting of [EC-(4β→8)]₉-EC, [EC-(4β→8)]₈-EC-(4β→6)-EC,[EC-(4β→8)]₈-EC-(4β→8)-C, and [EC-(4β→8)]₈-EC-(4β→6)-C.
 206. Thecomposition as claimed in claim 98 wherein the compound is selected fromthe group consisting of a trimer selected from the group consisting of[EC-(4β→8)]₂-EC, [EC-(4β→8)]₂-C and [EC-(4β→6)]₂-EC, a tetramer selectedfrom the group consisting of [EC-(4β→8)]₃-EC, [EC-(4β→8)]₃-C and[EC-(4β→8)]₂-EC-(4β→6)-C, a pentamer selected from the group consistingof [EC-(4β→8)]₄-EC, [EC-(4β→8)]₃-EC-(4β→6)-EC, [EC-(4β→8)]₃-EC-(4β→8)-Cand [EC-(4β→8)]₃-EC-(4β→6)-C, a hexamer selected from the groupconsisting of [EC-(4β→8)]₅-EC, [EC-(4β→8)]₄-EC-(4β→6)-EC,[EC-(4β→8)]₄-EC-(4β→8)-C, and [EC-(4β→8)]₄-EC-(4β→6)-C, a heptamerselected from the group consisting of [EC-(4β→8)]₆-EC,[EC-(4β→8)]₅-EC-(4β→6)-EC, [EC-(4β→8)]₅-EC-(4β→8)-C, and[EC-(4β→8)]₅-EC-(4β→6)-C, an octamer selected from the group consistingof [EC-(4β→8)]₇-EC, [EC-(4β→8)]₆-EC-(4β→6)-EC, [EC-(4β→8)]₆-EC-(4β→8)-C,and [EC-(4β→8)]₆-EC-(4β→6)-C, a nonamer selected from the groupconsisting of [EC-(4β→8)]₈-EC, [EC-(4β→8)]₇-EC-(4β→6)-EC,[EC-(4β→8)]₇-EC-(4β→8)-C, and [EC-(4β→8)]₇-EC-(4β→6)-C, a decamerselected from the group consisting of [EC-(4β→8)]₉-EC,[EC-(4β→8)]₈-EC-(4β→6)-EC, [EC-(4β→8)]₈-EC-(4β→8)-C, and [EC-(4β438)]₈-EC-(4β→6)-C, an undecamer selected from the group consisting of[EC-(4β→8)]₁₀-EC, [EC-(4β→8)]₉-EC-(4β→6)-EC, [EC-(4β→8)]₉-EC-(4β→8)-C,and [EC-(4β→8)]₉-EC-(4β→6)-C, and a dodecamer selected from the groupconsisting of [EC-(4β→8)]₁₁-EC, [EC-(4β→8)]₁₀-EC-(4β→3-6)-EC,[EC-(4β→8)]₁₀-EC-(4β→8)-C, and [EC-(4β→8)]₁₀-EC-(4β→6)-C.
 207. Thecomposition as claimed in claim 105 wherein the compound is selectedfrom the group consisting of a pentamer selected from the groupconsisting of [EC-(4β→8)]₄-EC, [EC-(4β→8)]₃-EC-(4β→6)-EC,[EC-(4β→8)]₃-EC-(4β→8)-C and [EC-(4β→8)]₃-EC-(4β→6)-C.
 208. Thecomposition as claimed in claim 119 wherein the compound is selectedfrom the group consisting of a trimer selected from the group consistingof [EC-(4β→8)]₂-EC, [EC-(4β→8)]₂-C and [EC-(4β→6)]₂-EC, a tetramerselected from the group consisting of [EC-(4β→8)]₃-EC, [EC-(4β→8)]₃-Cand [EC-(4β→8)]₂-EC-(4β→6)-C, a pentamer selected from the groupconsisting of [EC-(4β→8)]₄-EC, [EC-(4β→8)]₃-EC-(4β→6)-EC,[EC-(4β→8)]₃-EC-(4β43 8)-C and [EC-(4β→8)]₃-EC-(4β→6)-C, a hexamerselected from the group consisting of [EC-(4β→8)]₅-EC,[EC-(4β→8)]₄-EC-(4β→6)-EC, [EC-(4β→8)]₄-EC-(4β→8)-C, and[EC-(4β→8)]₄-EC-(4β→6)-C, a heptamer selected from the group consistingof [EC-(4β→8)]₆-EC, [EC-(4β→8)]₅-EC-(4β→6)-EC, [EC-(4β→8)]₅-EC-(4β→8)-C,and [EC-(4β→8)]₅-EC-(4β→6)-C, an octamer selected from the groupconsisting of [EC-(4β→8)]₇-EC, [EC-(4β→8)]₆-EC-(4β→6)-EC,[EC-(4β→8)]₆-EC-(4β→8)-C, and [EC-(4β→8)]₆-EC-(4β→6)-C, a nonamerselected from the group consisting of [EC-(4β→8)]₈-EC,[EC-(4β→8)]₇-EC-(4β→6)-EC, [EC-(4β→8)]₇-EC-(4β→8)-C, and[EC-(4β→8)]₇-EC-(4β→6)-C, and a decamer selected from the groupconsisting of [EC-(4β→8)]₉-EC, [EC-(4β→8)]₈-EC-(4β→6)-EC,[EC-(4β→8)]₈-EC-(4β→8)-C, and [EC-(4β→8)]₈-EC-(4β→6)-C.